Aquaporin-5 Protein Is Selectively Reduced in Rat Parotid Glands under Conditions of Fasting or a Liquid Diet

Abstract

Aquaporin-5 (AQP5) water channel, transmembrane protein 16A (TMEM16A) Ca²⁺-activated Cl^- channel, and Na⁺-K⁺-2Cl^- cotransporter (NKCC1) are membrane proteins on salivary gland acinar cells that function in watery saliva secretion. We examined their expression changes in rat parotid glands under reduced mastication. Rats were either fed regular chow as a control group, fasted for 48 hr or fed a liquid diet for 48 hr or 1 week to reduce mastication. The parotid glands were then resected to analyze the protein and mRNA levels by immunofluorescence, immunoblotting, and reverse-transcription quantitative PCR (RT-qPCR). AQP5 protein was significantly decreased in both liquid diet groups and the fasting group but its mRNA levels showed no apparent changes compared with the control group. The protein and mRNA levels of TMEM16A and NKCC1 showed no significant changes between any of the groups other than an increase in NKCC1 mRNA in the 1-week liquid diet group. These results suggest that reduced mastication may increase the AQP5 protein degradation, but not that of other membrane proteins necessary for saliva secretion.

Keywords: AQP5; fasting; liquid diet; parotid gland.

Fig. 1.

Immunofluorescence microscopy of AQP5, TMEM16A, and NKCC1 in the parotid glands of rats under liquid diet or fasting for 48 hr. Frozen sections of rat parotid glands from control, liquid diet, and fasting groups were labeled with anti-AQP5, anti-TMEM16A, or anti-NKCC1 antibodies. Representative images are shown. The exposure time for each antibody was fixed for the three groups.

Fig. 2.

Quantitative immunoblotting analysis of AQP5, TMEM16A, and NKCC1 in the parotid glands of rats under liquid diet or fasting for 48 hr. Immunoblotting signals for AQP5 (A), TMEM16A (B), NKCC1 (C), and β-actin are shown. Each band was quantified using NIH Image Software and the ratio of each protein to β-actin was then calculated. All results are the mean values ± SEM. Comparisons with the control group were performed using the two-tailed Dunnett’s test. *P < 0.05 from the control.

Fig. 3.

RT-qPCR analysis of AQP5, TMEM16A, and NKCC1 in the parotid glands of rats under liquid diet or fasting for 48 hr. Each mRNA in the liquid diet and fasting group is expressed as an n-fold difference relative to the control group (mean ± SEM). A: AQP5. B: TMEM16A. C: NKCC1. Comparisons with the control group were conducted using the two-tailed Dunnett’s test. *P < 0.05 from the control.

Fig. 4.

Immunofluorescence microscopy of AQP5, TMEM16A, and NKCC1 in the parotid glands of rats under liquid diet for one week. Frozen sections of rat parotid glands from control and liquid diet groups were labeled with anti-AQP5, anti-TMEM16A, or anti-NKCC1 antibodies. Representative images are shown. The exposure time for each antibody was fixed for the two groups.

Fig. 5.

Quantitative immunoblotting analysis of AQP5, TMEM16A, and NKCC1 in the parotid glands of rats under liquid diet for one week. Immunoblotting signals for AQP5 (A), TMEM16A (B), NKCC1 (C), and β-actin are shown. Each band was quantified using NIH Image Software and the ratio of each protein to β-actin was then calculated. All results are the mean values ± SEM. Comparisons were performed using a two-tailed unpaired t-test. *P < 0.05 from the control.

Fig. 6.

RT-qPCR analysis of AQP5, TMEM16A, and NKCC1 in the parotid glands of rats under liquid diet for one week. Each mRNA in the liquid diet group is expressed as an n-fold difference relative to the control group (mean ± SEM). A: AQP5. B: TMEM16A. C: NKCC1. Comparisons were conducted using a two-tailed, unpaired t-test. *P < 0.05 from the control.