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Age-related Decreases in the Response of Aquaporin-5 to Acetylcholine in Rat Parotid Glands

Department of Pharmacology, Tokushima University School of Dentistry, Kuramoto-cho, Tokushima 770-8504, Japan;

View larger version (38K): [in a new window]   Figure 1. Western blots for AQP5 in the homogenate and M3-muscarinic receptors and Gq/11 protein in basolateral membrane of parotid tissues. Parotid slices from 10-week-old (Young) or 115-week-old (Old) rats were incubated for 10 min at 37°C in the absence (1, 3, 5, 6) or presence (2, 4) of 10 µM SNI-2011. The homogenate (40 µg of protein) (A) and basolateral membrane (40 µg of protein) (B,C) of parotid glands from young adult (1, 2, 5) and senescent rats (3, 4, 6) were subjected to immunoblot analysis with antibodies (1:1500 dilution) to AQP5 (A), M3-mAChR (B), and Gq/11 (C).

View larger version (33K): [in a new window]   Figure 2. Effects of acetylcholine and epinephrine on AQP5 levels in the apical plasma membrane of parotid tissues from senescent and young adult rats. (A) Parotid slices from 10-week-old (Young) or 115-week-old (Old) rats were incubated for 15 sec at 37°C in the absence (C) or presence (ACh) of 1 µM acetylcholine and 1 µM eserine or incubated for 1 min at 37°C in the absence (C) or presence (Epi) of 10 µM epinephrine. The apical plasma membrane fraction (5 µg of protein) was subjected to immunoblot analysis with anti-AQP5 antibody. (B) Immunoblots were subjected to densitometric analysis, and the amount of AQP5 was expressed as a percentage of the control cell values. Data are expressed as means ± SE of 6 independent experiments. **P < 0.01, ***P < 0.001 vs. the value of the control cells.

View larger version (40K): [in a new window]   Figure 3. Time course of the effects of SNI-2011 and acetylcholine on the amount of AQP5 in the apical plasma membrane of parotid tissues from young adult and senescent rats. (A) Parotid slices from 10-week-old (Young) or 115-week-old (Old) rats were incubated at 37°C for 0, 1, 3, 10, or 30 min (lanes 1 to 5, respectively) in the presence of 10 µM SNI-2011. The apical plasma membrane fraction (5 µg of protein) was then subjected to immunoblot analysis with antibodies to AQP5. (B) Immunoblots were subjected to densitometric analysis, and the amount of AQP5 in 10-week-old ( O ) or 115-week-old ( • , solid line) rats was expressed as a percentage of the value for respective control tissues. Parotid slices from 115-week-old ( • , dotted line) rats were incubated at 37°C for 0, 0.25, 1, 3, or 10 min in the presence of 1 µM acetylcholine and 1 µM eserine. Data are means ± SE of values from 3 independent experiments performed with tissues from 3 different rats. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the control value.

View larger version (30K): [in a new window]   Figure 4. Effects of acetylcholine, epinephrine, and SNI-2011 on NOS activity in isolated parotid acinar cells from young adult or senescent rats. The isolated parotid acinar cells from 10-week-old (Young) or 115-week-old (Old) rats were pre-incubated with DAF-2/DA. Cells suspended with Krebs-Ringer bicarbonate solution were incubated with 1 µM acetylcholine(ACh) (A), 1 µM epinephrine (Epi) (B), and 10 µM SNI-2011 (C). The traces are representative of 3 independent experiments. Acetylcholine, epinephrine, and SNI-2011 were applied as indicated by the arrow. **P < 0.01, ***P < 0.001 vs. the control value.