Simultaneous monitoring of Ca2+ response and salivary secretion in rat submandibular gland in live animals

[Speaker] Akihiro Nezu:1
[Co-author] Takao Morita:2, Akihiko Tanimura:1
1:Department of Pharmacology, School of Dentistry, Health Sciences University of Hokkaido, Japan, 2:Department of Biochemistry, The Nippon Dental University School of Life Dentistry at Niigata, Japan

[Background] Effects of Ca2+ responses on the activation of ion channels have been studied extensively, while the Ca2+ responses during the salivary secretion are still unknown. Thus, we do not even know the intracellular Ca2+ concentration ([Ca2+]i) required for inducing fluid secretion. To address these questions, we developed a method for real time imaging of Ca2+ responses with simultaneous monitoring of salivary fluid secretion in live animal. [Methods] We expressed the genetically encoded Ca2+ indicator, YC-Nano50, following gene transfer via retrograde ductal injection of adenovirus vectors through the orifice of rat submandibular gland (SMG). To perform Ca2+ imaging in the YC-Nano50-expressing SMG, we used the multi-purpose zoom fluorescence microscope. Salivary secretion from SMG was measured using the fiber optic pressure sensor. [Results] An intravenous infusion of acetylcholine (ACh) increased [Ca2+]i throughout the SMG. ACh-induced Ca2+ responses and salivary flow rate in SMGs were increased in a dose-dependent manner (720 nmol/min). The apparent ED50 values for ACh stimulation on [Ca2+]i rise and salivary secretion were 74 and 118 nmol/min, respectively. Repetitive infusion of ACh induced similar levels of salivary secretion, associated with a similar rise in [Ca2+]i. Simultaneous monitoring of Ca2+ response and salivary secretion revealed a clear time lag between the onset of rise in [Ca2+]i and that of salivary secretion. The time lag with the lowest dose of ACh (30 nmol/min) was 106 s, which shortened to 19 s with the dose used for maximum salivary secretion (360 nmol/min). Based on the assumption that the resting [Ca2+]i in SMGs is 37 nM, the estimated [Ca2+]i at the onset of salivary secretions were 57 nM, irrespective of ACh dose. These results indicate that low [Ca2+]i is sufficient to trigger fluid secretion in the rat SMG in vivo . Interestingly, we found that low doses of ACh (< 120 nmol/min) induced Ca2+ oscillations with tissue-wide synchronization in SMG. These Ca2+ oscillations were associated with the oscillatory changes in blood flow. [Conclusions] We demonstrate Ca2+ responses during ACh-induced salivary secretions using a novel in vivo simultaneous monitoring of Ca2+ and salivary secretions.
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