Korean J Physiol Pharmacol.  2015 May;19(3):249-255. 10.4196/kjpp.2015.19.3.249.

Ca2+ is a Regulator of the WNK/OSR1/NKCC Pathway in a Human Salivary Gland Cell Line

• Park S
• Ku SK
• Ji HW
• Choi JH
• Shin DM

• KMID: 1791426
• DOI: http://doi.org/10.4196/kjpp.2015.19.3.249

Abstract

Wnk kinase maintains cell volume, regulating various transporters such as sodium-chloride cotransporter, potassium-chloride cotransporter, and sodium-potassium-chloride cotransporter 1 (NKCC1) through the phosphorylation of oxidative stress responsive kinase 1 (OSR1) and STE20/SPS1-related proline/alanine-rich kinase (SPAK). However, the activating mechanism of Wnk kinase in specific tissues and specific conditions is broadly unclear. In the present study, we used a human salivary gland (HSG) cell line as a model and showed that Ca2+ may have a role in regulating Wnk kinase in the HSG cell line. Through this study, we found that the HSG cell line expressed molecules participating in the WNK-OSR1-NKCC pathway, such as Wnk1, Wnk4, OSR1, SPAK, and NKCC1. The HSG cell line showed an intracellular Ca2+ concentration ([Ca2+]i) increase in response to hypotonic stimulation, and the response was synchronized with the phosphorylation of OSR1. Interestingly, when we inhibited the hypotonically induced [Ca2+]i increase with nonspecific Ca2+ channel blockers such as 2-aminoethoxydiphenyl borate, gadolinium, and lanthanum, the phosphorylated OSR1 level was also diminished. Moreover, a cyclopiazonic acid-induced passive [Ca2+]i elevation was evoked by the phosphorylation of OSR1, and the amount of phosphorylated OSR1 decreased when the cells were treated with BAPTA, a Ca2+ chelator. Finally, through that process, NKCC1 activity also decreased to maintain the cell volume in the HSG cell line. These results indicate that Ca2+ may regulate the WNK-OSR1 pathway and NKCC1 activity in the HSG cell line. This is the first demonstration that indicates upstream Ca2+ regulation of the WNK-OSR1 pathway in intact cells.

Keyword

Ca2+ signaling; NKCC; OSR1; Salivary gland; WNK

MeSH Terms

Cell Line*
Cell Size
Gadolinium
Humans
Lanthanum
Oxidative Stress
Phosphorylation
Phosphotransferases
Salivary Glands*
Sodium Chloride Symporters
Sodium-Potassium-Chloride Symporters
Gadolinium
Lanthanum
Phosphotransferases
Sodium Chloride Symporters
Sodium-Potassium-Chloride Symporters

Figure

• 

  Fig. 1 Phosphorylated OSR1 induced by hypotonicity and mRNA expression in a HSG cell line and phosphorylation of OSR1 in an isolated salivary gland acinar cell. (A) RT-PCR confirmed the expression of Wnk1, Wnk4, OSR1, SPAK, and NKCC1 at mRNA level. GAPDH expression was used as a control. (B) A hypotonic solution (215 mOsm) induced an increase in phosphorylated OSR1 (p-OSR1) in the HSG cell line (upper panel) and p-OSR1 in the parotid gland (PG) and submandibular gland (SMG) from ICR mice (lower panel). (C) The p-OSR1 level in the HSG cell line was normalized with the total OSR1 (right panel). HSG cell line n=3, mouse salivary gland acinar cell n=1, *p<0.05.

• 

  Fig. 2 Hypotonic stress-induced intracellular Ca2+ increases. The intracellular Ca2+ concentration ([Ca2+]i) was shown as the Fura2 intensity ratio of A340/A380. (A) A [Ca2+]i increase was observed when the cells were treated with a hypotonic solution, and the increased [Ca2+]i was diminished after the removal of extracellular Ca2+. (B) Pretreatment with 2APB, Gd3+, and La3+ reduced the [Ca2+]i induced by the hypotonic stimulation, and after washout of the blockers, the hypotonic stress-induced [Ca2+]i was recapitulated. (C) 4α-PDD evoked an increase in the [Ca2+]i. All the graphs show a representative trace, n=20~30 cells.

• 

  Fig. 3 Phosphorylation of OSR1 with various Ca2+ signaling-related drugs. (A) Pretreatment with 25 µM BAPTA/AM for 20 min followed by treatment with a hypotonic solution (215 mOsm) for 10 min. The fold change of the p-OSR1/OSR1 between control and BAPTA-loaded cells was statistically significant in the hypotonic stimulation. (B) Before hypotonic stimulation, each of the blockers was incubated for 20 min at 37℃, and then the cells were treated with the hypotonic solution for 10 min. 2APB 100 µM, RR 10 µM, Gd3+ 10 µM, and La3+ 10 µM was applied. (C, D) Up-regulated p-OSR1 was observed with both 25 µM CPA and 10 µM 4α-PDD applied in an isotonic solution (310 mOsm) *p<0.05. n=3 each.

• 

  Fig. 4 Down-regulation of NKCC activity is induced by hypotonic stress in a HSG cell line. Intracellular pH indicated by the BCECF signal ratio. (A) A decline in the rate after bumetanide (100 µM) treatment was decreased in an isotonic solution (310 mOsm); however, (B) after hypotonic stimulation (215 mOsm), the degree of the decline in the rate was reduced. (C) Chelating [Ca2+]i with 25 µM BAPTA-AM treatment restored the bumetanide-sensitive modification of the BCECF ratio. (A~C) The narrow black line represents a trend line indicating the slope of the pH declination. The alteration of the slope by treatment by 100 µM bumetanide is emphasized in the box. (D) The shifting slope of the declination was expressed by the fold change. (E) Schematic diagram that shows role of NH4Cl in the intracellular pH modification. All the traces are representative graphs, with n=25~35 cells in each experiment. *p<0.05.

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