Intravital Two-photon Imaging of Ca2+ signaling in Secretory Organs of Yellow Cameleon Transgenic Mice

Kai Jin,Hisayo Sakaguchi,Erina Oonishi,Shigeru Nakamura,Toshihiro Imada,Kazuo Tsubota,Takahiro Adachi,Michiko Shibuya,Yusuke Izuta

doi:10.1038/s41598-018-34347-1

Kai Jin, Hisayo Sakaguchi + Show 7 more

Open Access

https://doi.org/10.1038/s41598-018-34347-1

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Abstract

Intracellular calcium ([Ca2+]i) signaling regulates physiological functions in most cells. In secretory organs, such as the pancreas, salivary gland, and lacrimal gland (LG), [Ca2+]i elevation in acinar cells triggers fluid secretion, which plays vital roles in the maintenance of functional health across the life-course. It is important to understand the secretory mechanism of secretory organs, but lack of analytic systems available for living animals limits the scope of research to gain deeper insights into the precise mechanism of secretion. We established an intravital imaging system for specific cell types of secretory organs to monitor the [Ca2+]i changes using mouse line expressing Yellow Cameleon 3.60, a genetically encoded Ca2+ indicator. Elevation of [Ca2+]i in specific cell types of secretory organs could be monitored after cholinergic stimulation ex vivo and intravitally. We found that a marked attenuation of LG [Ca2+]i response to cholinergic stimulation was induced under pathological conditions by postganglionic denervation. Intravital Ca2+ imaging in secretory organs will broaden our understanding of the cellular mechanisms in animal models of secretory diseases.

Highlights

Intracellular Ca2+ signaling plays important roles in regulating a wide variety of cellular physiological processes[1–3]
In secretory organs, such as the pancreas, salivary gland (SG), and lacrimal gland (LG), [Ca2+]i elevation in the secretory acinar cells is the key trigger for secretion of a mixture of water and proteins synthesized in the acinar cells[3–5]
We initially evaluated the changes in fluorescence resonance energy transfer (FRET) ratios induced by the calcium ionophore ionomycin; cyclopiazonic acid (CPA) which is an inhibitor of the endoplasmic reticulum Ca2+ ATPase pump[23] and the muscarinic acetylcholine receptors (mAChRs) agonist acetylcholine (ACh) using isolated pancreases from YC3.60 transgenic mice by using our ex vivo imaging system

Summary

Introduction

Intracellular Ca2+ signaling plays important roles in regulating a wide variety of cellular physiological processes[1–3]. Intracellular Ca2+ concentration ([Ca2+]i) is regulated by intracellular release from the endoplasmic reticulum store or influx through a variety of Ca2+ channels in response to stimulation by neurotransmitters and a variety of hormones[1,2]. In secretory organs, such as the pancreas, salivary gland (SG), and lacrimal gland (LG), [Ca2+]i elevation in the secretory acinar cells is the key trigger for secretion of a mixture of water and proteins synthesized in the acinar cells[3–5]. Synthetic Ca2+ indicators, such as Fura-2, Fluo[4] and Indo 1, have been extensively used for monitoring intracellular Ca2+ signaling[9–11] These synthetic Ca2+ indicators exhibit high sensitivity to Ca2+ and rapid response kinetics, the disadvantages include limitation of loading to specific cell types within an intact tissue and insufficient intracellular retention[10]. Photostability but the advantages of them are less sensitive to motion artifact and expression level differences because of rationing of two fluorescent proteins[13,14]

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