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Am J Physiol Renal Physiol 294: F788-F794, 2008. First published January 16, 2008; doi:10.1152/ajprenal.00553.2007
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Stanniocalcin-1 secretion and receptor regulation in kidney cells

Olga Sazonova, Kathi A. James, Christopher R. McCudden, Daniel Segal, Asghar Talebian, and Graham F. Wagner

Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada

Submitted 20 November 2007 ; accepted in final form 14 January 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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.

Madin-Darby canine kidney; inner medullary collecting duct-3; ligand

THE MAMMALIAN stanniocalcin-1 (STC-1) gene is widely expressed and mRNA levels are highest in the ovary, followed by kidney, adrenal, prostate, and heart (9). Within the kidneys, STC-1 is involved in transport-related processes, organogenesis, and mitochondrial function. In the rat, the transport-related effects are thought to involve blood-borne hormone that is delivered to nephrons attached to a soluble binding protein. The complex is filtered out by the glomerulus and targets luminal membranes of the proximal tubule S3 segment to increase phosphate transport (13). This has been demonstrated in vivo in adult rats, where bolus injections of human STC-1 increased renal phosphate reabsorption through a doubling in proximal tubular sodium-phosphate cotransporter activity without concomitant effects on serum levels of calcium or phosphate (22, 33). However, chronic elevations in serum STC-1 in transgenic mice can significantly alter serum levels of calcium and/or phosphate (8, 31).

A local STC-1 signaling pathway is also operative within the kidneys. In rodent kidney models, gene expression is confined to collecting duct cells and expression levels are highest in embryos and neonates. In embryos, STC-1 is heavily targeted to the nephrogenic zone, suggestive of a role in early development (28). STC-1 appears to be especially important to kidney function in neonates, where expression levels are 10-fold higher than in adults (6). There, collecting duct-derived STC-1 is targeted to upstream nephron segments such as the proximal tubules, where it could be aiding the higher nutritional requirements of newborns for inorganic phosphate (6).

In adult rodents, collecting duct cell-derived STC-1 is targeted locally to thick ascending limb and distal convoluted tubule cells and back onto collecting duct cells. All targeted cells possess high-affinity receptors on the plasma membranes and both mitochondrial membranes (inner and outer). These aid in the sequestration of STC-1 within the matrix to the extent that mitochondria contain high levels of STC-1 by both Western blotting and immunocytochemistry (16). Within the matrix, STC-1 increases electron transport chain activity while uncoupling oxidative phosphorylation, the consequence of which is increased mitochondrial calcium uptake (7). STC-1 also has concentration-dependent effects on oxygen consumption as shown by the mouse inner medullary collecting duct cell line IMCD-3 (7). The respiratory effects of STC-1 have been observed in liver, muscle, and kidney mitochondria and/or cells and hence may be part of a general mechanism by which the hormone is cytoprotective under conditions of stress. Evidence of this is derived from other studies showing that STC-1 has antiapoptotic effects in human neuronal cells under hypoxic stress, in part through reductions in intracellular calcium (37). Similarly, STC-1 plays a yet undetermined role in the adjustment of Madin-Darby canine kidney (MDCK) cells to hypertonicity. After exposure to impermeant solutes such as NaCl, STC-1 mRNA levels increase markedly (27). Moreover, as in the case of IMCD-3 cells, MDCK cells possess high-affinity STC-1 receptors on both their plasma and mitochondrial membranes (7).

In different ways, the IMCD-3 and MDCK renal cell lines have proven useful in studies of STC-1 function. The IMCD-3 line does not produce measurable amounts of STC-1 but is highly responsive to exogenous hormone (7). MDCK cells, on the other hand, have measurable levels of production (27) and hence are ideally suited for studies on regulated secretion. The aim of the present study, therefore, was to investigate the regulation of STC-1 secretion by MDCK cells under normal and hypertonic conditions. We also sought to examine the effects of other hormones known to influence nephron cell function, as well as the effects of activators/inhibitors of classic signal transduction pathways on STC-1 secretion. Finally, the MDCK and IMCD-3 lines afforded us the opportunity to examine STC-1 receptor regulation in response to changes in ligand concentration.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cells, reagents, and experimental paradigms. The MDCK-1 clonal cell line was provided by Dr. Tony D'Souza (Dept. of Physiology and Pharmacology, University of Western Ontario). Cells were maintained in DMEM/F-12 medium containing 10% fetal bovine serum (FBS; GIBCO BRL) and were grown to confluence in 24-well plates before each experiment. For experiments involving hormones, the medium was switched to serum-free DMEM/F-12 containing 0.1% bovine serum albumin. IMCD-3 cells were provided by Dr. Norman Rosenblum (Hospital for Sick Children, Toronto, Canada). IMCD-3 cells were grown to confluence in chamber slides, 24-well plates, or T75 flasks and incubated at 37°C in DMEM containing 5% FBS.

To determine whether STC-1 was secreted in vectoral fashion, cells were plated to confluence on transwell inserts and maintained thus for up to 6 days. The media in both chambers were replaced on a daily basis and analyzed for STC-1 content. The study was repeated three times on quadruplicate wells of cells.

For experiments testing the effects of hormones, drugs, and electrolytes on STC-1 secretion, additions to cultured cells were made from 100-fold concentrates. In some cases, media were prepared beforehand containing the desired concentrations and added directly to the cells. Angiotensin II, atrial natriuretic factor (ANF), antidiuretic hormone (ADH), ionomycin, and the phorbol ester TPA were obtained from Sigma Chemicals (St. Louis, Mo.). The calcimetics NPS R-467 and S-467 were gifts from NPS Pharmaceuticals (Salt Lake City, UT). Biologically active recombinant human STC-1 (hSTC-1) was obtained from Human Genome Sciences (Rockville, MD) (38). Each of the above studies was repeated at least twice on triplicate wells of cells. Conditioned media STC-1 levels were monitored with a double-antibody human STC-1 radioimmunoassay that has already been characterized for specificity (20).

In situ ligand binding studies. To localize STC-1 binding sites at the histological level before and after hSTC-1 treatment, we conducted in situ ligand binding on confluent IMCD-3 cells plated on chamber slides (Lab-Tek II). Experimental cells were exposed to 500 nM hSTC for 1 h, whereas control cells received buffer alone. Cells were fixed in 4% paraformaldehyde in PBS before the detection of STC-1 biding sites as previously described (16). Binding sites on plated, confluent MDCK cells were detected in the same manner, following 24-h exposures to media containing normal (150 mM) and high concentrations (300 mM) of NaCl. Digital images were captured using a Zeiss AX10 microscope with a Retiga 1300 camera.

Measurements of STC-1 receptor levels. Receptors were quantified on confluent MDCK cells following 24-h exposures to increasing concentrations of NaCl or CaCl2. Cells were washed in serum-free media and fixed for 30 s in PBS containing 4% paraformaldehyde. After two rinses in PBS, the plated cells were assessed for receptor binding activity as previously described (16). Receptor levels were quantified on confluent IMCD-3 cells following 0.5- and 1-h exposures to media with or without 500 nM hSTC-1. Each study was repeated three times on quadruplicate wells of cells.

In additional experiments, receptor binding studies were conducted to monitor possible changes in IMCD-3 mitochondrial and plasma membrane receptor levels after exposure to hSTC-1. Cells were grown to confluence in T75 flasks and incubated with 500 nM hSTC-1 or control media for 1 h. The cells were washed and harvested, and plasma membrane and mitochondrial receptor levels were assessed as previously described (16). This study was repeated twice.

Statistical analysis. Data sets were analyzed using one-way analysis of variance (ANOVA) and, if necessary, Dunnet's multiple comparison test using GraphPad InStat software. Groups were considered significantly different if P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of permeant and nonpermeant ions on MDCK STC-1 secretion and receptor levels. Previous studies have shown that nonpermeant ions such as NaCl and raffinose have stimulatory effects on the levels of STC-1 mRNA in MDCK cells, whereas permeant ions such as urea do not (27). In agreement with these findings, additions of NaCl had concentration and time-dependent stimulatory effects on STC-1 release (Fig. 1A). A 24-h exposure to NaCl caused a threefold stimulation of STC-1 release, with the greatest effects being obtained with additions of 125 mM NaCl (P < 0.01; ANOVA and Dunnet's test). Higher concentrations of NaCl inhibited hormone release without any apparent effects on cell viability (Fig. 1A). Furthermore, both the appearance and viability of the cells was affected by additions of 250 mM NaCl and higher. The time course effects of NaCl are shown in Fig. 1B, where a statistically significant effect on secretion was evident by 2 h and persisted up to 48 h (P < 0.01–0.001; ANOVA and Dunnet's test).


Figure 1
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  		Fig. 1. Madin-Darby canine kidney (MDCK) cell secretory responses to NaCl and urea. A: stanniocalcin-1 (STC-1) secretion after a 24-h exposure to progressively higher levels of NaCl-induced hypertonicity. B: time-course effects of an additional 50 mM of NaCl on STC-1 secretion. C: STC-1 receptor levels after a 24-h exposure to progressively higher levels of NaCl-induced hypertonicity. D: STC-1 secretion after a 24-h exposure to progressively higher levels of urea-induced hyperosmolarity. Values are means ± SE; n = 4 wells per treatment in A–D. *P < 0.05; **P < 0.01; ***P < 0.001 (ANOVA and Dunnet's test).

 
Although increasing concentrations of NaCl produced stepwise increases in MDCK cell STC-1 secretion, this resulted in no corresponding changes, upward or downward, in STC-1 receptor levels (Fig. 1C). In contrast to the hypertonic effects of NaCl, the hyperosmolar effects of urea were inhibitory rather than stimulatory and were most evident at the highest concentrations employed (Fig. 1D).

Effects of CaCl2 on MDCK STC-1 secretion and receptor levels. Additions of CaCl2 had concentration-dependent inhibitory effects on STC-1 release between 1 and 10 mM, reducing STC-1 output by >50% at the highest concentrations employed (Fig. 2A; P < 0.01; ANOVA and Dunnet's test). In contrast, as hormone secretion was suppressed by increasing calcium concentrations, STC-1 receptor levels were correspondingly increased (Fig. 2B; P < 0.05–0.01; ANOVA and Dunnet's test), to the extent that linear regression (Fig. 2C) revealed a significant inverse relationship between ligand output and total cellular binding activity (R2 = 0.4; P < 0.02; ANOVA).


Figure 2
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  		Fig. 2. MDCK cell secretory responses to CaCl2 and vectoral STC-1 release. A: media STC-1 content after 24-h exposure to increasing concentrations of CaCl2. B: cellular STC-1 receptor levels after 24-h exposure to increasing concentrations of CaCl2. C: linear regression analysis of STC-1 ligand and receptor levels from the data in A and B. D: vectoral STC-1 secretion in untreated confluent monolayers plated in transwell inserts. Secretion was preferentially apical during the first 2 days. Values are means ± SE; n = 4 wells per treatment in A, B, and D. *P < 0.05; **P < 0.01 (ANOVA and Dunnet's test).

 
Vectoral STC-1 secretion. To examine vectoral secretion, MDCK cells were plated on transwell inserts and grown to confluence. The media were then changed daily thereafter in both chambers for up to 6 days. When analyzed for hormone content, the medium bathing the apical surface of the monolayer had significantly higher STC-1 levels (P < 0.01; ANOVA and Dunnet's test) during the first 2 days, indicative of preferential, albeit not exclusive, apical release (Fig. 2D). From day 3 onward, however, hormone release occurred equally from both domains. As such, the evidence suggests there was no preference for vectoral STC-1 release in MDCK cells.

Effects of hormones and signal transduction pathway activators on STC-1 release. To identify the pathway involved in NaCl-regulated STC-1 release, we tested various hormones and signal transduction pathway activators for possible effects. Forskolin, even at high concentrations (1 µM), and ADH (10–8–10–12 M) had no discernable effects on hormone output (results not shown), suggesting that the protein kinase A (PKA) pathway was not involved. ANF (10–7–10–11 M) and angiotensin II (10–7–10–8 M), operating through the protein kinase C (PKC) pathway, also had no effects (positive or negative) on either resting or NaCl-stimulated STC-1 release (results not shown).

To explore in more detail the inhibitory effects of added calcium, we tested calcimetics, ionomycin, and TPA were tested for their effects on hormone release. The calcimetic R-467 (10–5–10–7 M) and its enantiomer, S-467, had no discernable effects on STC-1 release, suggesting that the inhibitory effects of calcium were not mediated through calcium-sensing receptors (results not shown). Ionomycin, on the hand, inhibited STC-1 release and did so to a much greater extent than added CaCl2. Figure 3A shows the effects of increasing ionomycin levels (0.1–10 µM) on STC-1 release, with the highest levels inducing >90% inhibition of hormone output with no discernable effects on the appearance or viability of the cells. In contrast, a 24-h exposure to TPA (50 nM) stimulated hormone release, although not to the same extent as NaCl (Fig. 3B).


Figure 3
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  		Fig. 3. MDCK cell secretory responses to ionomycin and TPA. A: inhibitory effects of increasing ionomycin levels on basal and NaCl-stimulated STC-1 secretion after 24 h. B: comparative effects of ionomycin, TPA, and NaCl on STC-1 secretion. Values are means ± SE; n = 4 wells per treatment in A and B. *P < 0.05; **P < 0.01; ***P < 0.001 (ANOVA and Dunnet's test).

 
STC-1 receptor regulation in IMCD-3 cells. Treatment of IMCD-3 cells with 500 nM hSTC-1, a concentration previously shown to significantly increase respiration rate (7), produced no significant changes in total cellular receptor levels after 0.5- and 1.0-h exposures (Fig. 4A). Similarly, 500 nM hSTC-1 treatment also had no effect on membrane or mitochondrial receptor levels following a 1-h exposure (Fig. 4B).


Figure 4
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  		Fig. 4. Effects of recombinant human STC-1 (hSTC-1) treatment on mitochondria and membrane receptor levels in IMCD-3 cells. A: after treatment with 500 nM hSTC for 0.5 and 1.0 h, receptor levels were quantified directly on plated cells. B: after treatment with 500 nM hSTC for 1 h, receptor levels were quantified on isolated plasma membranes and mitochondria. There were no significant effects of STC-1 treatment on receptor levels in whole cells (A) or isolated plasma membranes and mitochondria (B). CTL, control. Values are means ± SE; n = 4 for A and B. P > 0.05 (Student's t-test).

 
In situ ligand binding studies. In situ ligand binding studies on untreated MDCK cells revealed that binding activity was evenly distributed throughout the cytoplasm (Fig. 5A). In contrast, in situ ligand binding on IMCD-3 cells produced a punctate pattern of staining (Fig. 5B) similar to that seen in paraffin sections of distal convoluted tubule and collecting duct cells (16) and possibly indicative of mitochondrial-sited receptors. In contrast, the nuclei in both cell types were notable by their lack of binding activity, although in isolated cases (~1%), nuclear binding was observed over isolated IMCD-3 cells (Fig. 5C).


Figure 5
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  		Fig. 5. In situ localization of STC-1 receptors in MDCK and IMCD-3 cells. A: in MDCK cells, STC-1 binding activity was equally distributed throughout the cytoplasm, with little or no binding seen over the nucleus (arrow). B: in IMCD-3 cells, binding activity was distributed in punctate fashion throughout the cytoplasm, possibly indicative of mitochondrial receptors. The nuclei were notable by their lack of binding activity (arrow). Inset (bottom left) is a staining control. C: in a small number (~1%) of IMCD-3 cells, binding activity was concentrated over the nucleus (arrow).

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The MDCK cell line has been used extensively for studies on survival responses to stress, particularly hypertonic stress. The cell line is distal tubular in origin and is variably described as having been descended from the thick ascending limb/distal convoluted tubule (12) or the collecting duct principal cell (19). The cell line is favored for studies on how nephron cells respond to hypertonicity in terms of the genes upregulated and the regulatory pathways involved. Nonpermeant ions such as NaCl induce the immediate early gene transcription factors Egr-1 and c-fos, as well as heat shock protein 70 (5). The production of osmolytes such as betaine and myo-inositol are upregulated to conserve cellular water (18, 35, 36), as are the levels of Na+-K+ ATPase to rid cells of unwanted sodium (1). Genes such as the myo-inositol transporter are essential for cell survival (14). Of particular interest to us was the fact that STC-1 was induced in response to NaCl-induced hypertonic stress, as much as eightfold following additions of 100–200 mM NaCl (27). A response of this magnitude was indicative of STC-1 having some importance in surviving the stress of hypertonicity. This was perhaps not surprising in view of the growing evidence that STC-1 has antiapoptotic effects, particularly in cells under stress.

Previous studies have shown that STC-1 has pronounced cytoprotective effects in human neuronal cells by delaying the onset of hypoxia-induced apoptosis (37). In the process, STC-1 enhanced phosphate uptake by the cells, thereby attenuating the rise in intracellular calcium levels (37). In further support of this notion, through the uncoupling of oxidative phosphorylation, STC-1 stimulates mitochondrial calcium import (7), which would similarly attenuate rising intracellular calcium, and since STC-1 receptors are also present on MDCK cell mitochondria (7), the ligand could be similarly involved there in attenuating rising cytosolic calcium. Earlier studies have shown that the response of these cells to hypertonicity involves activation of phospholipase C (PLC), inositol 1,4,5-trisphosphate (IP3), and PKC, all of which would increase intracellular calcium levels (29). Moreover, renal failure is associated with increased endothelin-1 (ET-1) and cell calcium overload in both ischemic and nephrotoxic models (23). Since it is under these very circumstances that STC-1 secretion is induced (and by TPA in the present study), its target and intent could very well be the mitochondria and the dampening of intracellular calcium levels. It should also be pointed out that free radical formation occurs hand in hand with oxidative phosphorylation. Thus, through its respiratory uncoupling effects, STC-1 may be attenuating cell damage through reductions in the levels of both intracellular calcium and oxygen free radicals.

Given the fact that the hypertonic response prompts PLC/IP3/PKC activation (29) and STC-1 secretion, a puzzling aspect of the present study was the inability of angiotensin II, a known stimulator of PKC and intracellular calcium levels (17, 21), to affect hormone release. Similarly, PKA activation has likewise been shown to increase intracellular calcium (4) and yet was entirely ineffective in the present study. Equally contradictory were the highly suppressive effects of ionomycin on both basal and NaCl-stimulated STC-1 release. It is worth noting, however, that in all of the studies cited above involving PKA and PKC activation, the resulting increases in intracellular calcium were not particularly large. What the findings collectively suggest, therefore, is that the hypertonicity-induced release of STC-1 is much more complex than currently appreciated and may involve one of the atypical PKCs that do not require calcium, such as the {zeta}, {iota}, and/or {lambda} isoforms.

The inability of calcium-sensing receptor agonists to affect MDCK cell STC-1 secretion were in contrast to their stimulatory effects in fish (24). So too were the highly suppressive effects of ionomycin, given that calcium channel activators such as A-23187 have been shown to stimulate hormone release in fish (32). However, it is also true that mammalian STC-1 does not regulate extracellular calcium levels as it does in fish (9). That being said, the inhibitory effects of high intracellular calcium on MDCK cell STC-1 release were in complete accordance with in vivo studies in the rat, where 1% calcium gluconate in the drinking water significantly reduced STC-1 mRNA levels in medullary collecting duct cells (6). In this respect and in their high rate of STC-1 production, the MDCK-1 line best models the collecting duct principal cell. That having been said, the underlying purpose behind the suppressive effects of calcium still remains to be established.

MDCK cells have also been used to study vectoral secretion of polypeptide hormones and the factors determining apical versus basolateral release. For instance, ET-1 is preferentially released basolaterally from cells grown on transwell inserts (26). Moreover, carbohydrate moieties can in some cases be a major determinant of vectoral release (25). A good example is erythropoietin, which is preferentially released by the apical domain. In the presence of tunicamycin, however, which blocks N-glycosylation, apical preference is lost (15). Similarly, microtubule disruptors can reverse an established pattern of vectoral secretion (15). In the present study, MDCK cells secreted STC-1 from both the apical and basolateral domains, in approximately equal proportions. In this respect they again resembled collecting duct principal cells in vivo. In neonate and adult mice, principal cells are the main site of STC-1 production and secretion (6, 34). The ligand is then targeted to adjacent upstream nephron segments (thick ascending limb, distal convoluted tubules) and back on to collecting duct cells (16). Whereas paracrine targeting to adjacent nephron segments requires that ligand release is basolateral, the autocrine targeting of collecting duct cells may also entail apical release for luminal targeting of cells further downstream. Histological evidence in support of this comes from the fact that collecting duct STC-1 receptors in mice (embryos and adults) are often preferentially sited on the apical membranes (Wagner GF, unpublished observations), as they sometimes are on the proximal straight tubules (13).

In the majority of cases, peptide receptors are downregulated upon ligand binding due to internalization of the hormone-receptor complex. In contrast, the withdrawal of ligand generally results in their upregulation. There are exceptions, of course, such as in the case of gonadotropin-releasing hormone receptors, which can be upregulated by cognate ligand binding (30). In the case of STC-1, changes in mammary gland nuclear receptor levels have been monitored in vivo, where they proved to be positively correlated with changes in plasma levels of the hormone (11). However, in vitro responses to changes in STC-1 levels have not been examined to date. It was with this in mind that receptor levels were monitored in MDCK and IMCD-3 cells, both of which have high-affinity receptors on the plasma and mitochondrial membranes (7). Conditions of endogenous ligand excess were examined first in MDCK cells exposed to NaCl-induced hypertonicity. However, despite a tripling of hormone levels, STC-1 receptor levels remained unchanged. Similar studies with the IMCD-3 line, in which 500 nM hSTC-1 was added directly to the cells, yielded essentially the same results: no changes in plasma membrane or mitochondrial receptor levels. What these findings suggest, first of all, is that the receptor is not downregulated by either short (0.5–1.0 h)- or long-term exposures (24 h) to the ligand. If this is indeed the case, then from a mechanistic standpoint the findings further imply that the receptor does not serve as a chaperone for the ligand as it traffics from the cell surface to the mitochondria. Instead, it would appear that there are independent receptor pools on the plasma and mitochondrial membranes that facilitate ligand passage from the cell surface into the mitochondria and that these pools remain relatively constant. Alternatively, we must also consider the possibility that the receptor does in fact shuttle between the cell surface and mitochondria but at a speed that precludes detection under the present experimental conditions. Finally, in contrast to the effects of ligand excess, inhibiting STC-1 secretion with high levels of calcium in the medium caused a significant upregulation in receptor levels. Although such a response typified that of most peptide hormone receptors, a possible direct effect of calcium on receptor half-life and/or rate of synthesis should not be ruled out.

In summary, MDCK cells responded to NaCl-induced hypertonicity with heightened secretory activity, whereas secretion was inhibited by high levels of intracellular calcium. The inhibitory effects of calcium on secretion were reminiscent of those produced by increased dietary calcium, which lowered STC-1 transcript levels in rat inner medullary collecting duct cells in vivo. Intriguingly, plasma membrane and mitochondrial STC-1 receptor levels were unchanged in MDCK and IMCD-3 cells after exposure to high ligand levels, suggesting that the receptor may not accompany STC-1 in its passage to the mitochondria. One important question that now needs to be addressed is the means by which STC-1 traverses the nephron cell cytoplasm and reaches the mitochondria.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
Support was provided by the Kidney Foundation of Canada and the Canadian Institutes of Health Research (to G. F. Wagner).


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. F. Wagner, Dept. of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, Univ. of Western Ontario, London, Ontario, Canada N6A 5C1 (e-mail: graham.wagner{at}schulich.uwo.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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