Abstract
In order to assess the contribution of the water channel aquaporin-5 (AQP5) to water transport by salivary gland acinar cells, we measured the cell volume and activation energy (E a) of diffusive water permeability in isolated parotid acinar cells obtained from AQP5-G103D mutant and their wild-type rats. Immunohistochemistry showed that there was no change induced by carbamylcholine (CCh; 1 μM) in the AQP5 detected in the acinar cells in the wild-type rat. Acinar cells from mutant rats, producing low levels of AQP5 in the apical membrane, showed a minimal increase in the AQP5 due to the CCh. In the wild-type rat, CCh caused a transient swelling of the acinus, followed by a rapid agonist-induced cell shrinkage, reaching a plateau at 30 s. In the mutant rat, the acinus did not swell by CCh challenge, and the agonist-induced cell shrinkage was delayed by 8 s, reaching a transient minimum at around 1 min, and recovered spontaneously even though CCh was persistently present. In the unstimulated wild-type acinar cells, E a was 3.4 ± 0.6 kcal mol−1 and showed no detectable change after CCh stimulation. In the unstimulated mutant acinar cells, high E a value (5.9 ± 0.1 kcal mol−1) was detected and showed a minimal decrease after CCh stimulation (5.0 ± 0.3 kcal mol−1). These results suggested that AQP5 was the main pathway for water transport in the acinar cells and that it was responsible for the rapid agonist-induced acinar cell shrinkage and also necessary to keep the acinar cell volume reduced during the steady secretion in the wild-type rat.
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Abbreviations
- AQP5:
-
Aquaporin-5
- CCh:
-
Carbamylcholine
- E a :
-
Activation energy
- Gd-DTPA:
-
Gadolinium-diethylene triamine-N,N,N′,N″,N″-pentaacetic acid
- KRB:
-
Krebs–Ringer bicarbonate
- P d :
-
Diffusive membrane permeability
- SD:
-
Sprague-Dawley
- SMG:
-
Submandibular salivary gland
- T1 :
-
Spin-lattice relaxation time
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Acknowledgments
The authors would like to thank Dr. T. Hasegawa (UTGS) for his analysis of the AQP5 gene, Dr. K. Manaka (Research Support Center, DMUSM) for his support of the confocal imaging, and also Ms. Y. Imaizumi-Ohashi and Ms. M. Yokoi-Hayakawa (DMUSM) for their technical assistance. A part of this study was supported by grants from the Ministry of Education, Science, and Culture of Japan.
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Supplementary Figure 1
Amylase release from isolated rat parotid acinar cells upon isoproterenol stimulation. Isolated acinar cells were stimulated by isoproterenol (1 μM) for 20 min. Amylase release was expressed as the percentage of the total amylase activity. Solid column without isoproterenol stimulation; clear column after isoproterenol stimulation. Values are means ± SEM from three independent experiments. Results were analyzed by Student’s t test: *P < 0.05. (PDF 16 kb)
Supplementary Figure 2
Dependency of fraction of AQP5 on the apparent E a, P d, and P f. This is a simulation of apparent E a, P d, and P f based on an assumption of two water transport pathways. One pathway was transported through AQP5 (P d = 1 × 10−3 cm s−1, E a = 3 kcal mol−1), and the other was transported through the lipid membrane per se (P d = 1 × 10−3 cm s−1, E a = 12.75 kcal mol−1) both at 25 °C. The apparent E a values were calculated from the Arrhenius plot simulated at temperatures of 5, 15, 25, and 30 °C. A red circle shows an E a (6 kcal mol−1) which was obtained at a fraction of AQP5 of 15 %. The apparent P f was estimated on the assumption of the P f/P d ratios of AQP5, and the lipid membrane were 5–10 and 1, respectively. (PDF 14 kb)
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Satoh, K., Seo, Y., Matsuo, S. et al. Roles of AQP5/AQP5-G103D in carbamylcholine-induced volume decrease and in reduction of the activation energy for water transport by rat parotid acinar cells. Pflugers Arch - Eur J Physiol 464, 375–389 (2012). https://doi.org/10.1007/s00424-012-1141-8
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DOI: https://doi.org/10.1007/s00424-012-1141-8