Basic Study
Copyright ©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Feb 7, 2015; 21(5): 1444-1456
Published online Feb 7, 2015. doi: 10.3748/wjg.v21.i5.1444
Mechanism involved in Danshen-induced fluid secretion in salivary glands
Fei Wei, Mu-Xin Wei, Masataka Murakami
Fei Wei, Division of Physiological Sciences, School of Life Science, Graduate University for Advanced Studies, Okazaki 444-8585, Japan
Mu-Xin Wei, Department of Traditional Chinese Medicine, The first Affiliated Hospital with Nanjing Medical University, Nanjing 210029, Jiangsu Province, China
Masataka Murakami, Division of Cellular Physiology, National Institute for Physiological Sciences, National Institutes of Natural Sciences, Okazaki 444-8787, Japan
Author contributions: Wei F was involved in the whole experiment and in preparing the manuscript; Wei MX was involved in choosing the Danshen employed; Murakami M provided the experimental system, including reagents and analytical tools, and was also involved in the design of the whole experiment and editing the manuscript.
Supported by Grants from The National Institute for Physiological Sciences and Graduate University for Advanced Studies.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Masataka Murakami, Associate Professor, Division of Cellular Physiology, National Institute for Physiological Sciences, National Institutes of Natural Sciences, 5-1 Higashiyama, Myoudaiji-cho, Okazaki 444-8787, Japan. masataka@nips.ac.jp
Telephone: +81-564-595268 Fax: +81-564-595269
Received: July 23, 2014
Peer-review started: July 24, 2014
First decision: September 15, 2014
Revised: September 29, 2014
Accepted: November 19, 2014
Article in press: November 19, 2014
Published online: February 7, 2015
Processing time: 201 Days and 0.4 Hours
Abstract

AIM: Danshen’s capability to induce salivary fluid secretion and its mechanisms were studied to determine if it could improve xerostomia.

METHODS: Submandibular glands were isolated from male Wistar rats under systemic anesthesia with pentobarbital sodium. The artery was cannulated and vascularly perfused at a constant rate. The excretory duct was also cannulated and the secreted saliva was weighed in a cup on an electronic balance. The weight of the accumulated saliva was measured every 3 s and the salivary flow rate was calculated. In addition, the arterio-venous difference in the partial oxygen pressure was measured as an indicator of oxygen consumption. In order to assess the mechanism involved in Danshen-induced fluid secretion, either ouabain (an inhibitor of Na+/K+ ATPase) or bumetanide (an inhibitor of NKCC1) was additionally applied during the Danshen stimulation. In order to examine the involvement of the main membrane receptors, atropine was added to block the M3 muscarinic receptors, or phentolamine was added to block the α1 adrenergic receptors. In order to examine the requirement for extracellular Ca2+, Danshen was applied during the perfusion with nominal Ca2+ free solution.

RESULTS: Although Danshen induced salivary fluid secretion, 88.7 ± 12.8 μL/g-min, n = 9, (the highest value around 20 min from start of DS perfusion was significantly high vs 32.5 ± 5.3 μL/g-min by carbamylcholine, P = 0.00093 by t-test) in the submandibular glands, the time course of that secretion differed from that induced by carbamylcholine. There was a latency associated with the fluid secretion induced by Danshen, followed by a gradual increase in the secretion to its highest value, which was in turn followed by a slow decline to a near zero level. The application of either ouabain or bumetanide inhibited the fluid secretion by 85% or 93%, and suppressed the oxygen consumption by 49% or 66%, respectively. These results indicated that Danshen activates Na+/K+ ATPase and NKCC1 to maintain Cl- release and K+ release for fluid secretion. Neither atropine or phentolamine inhibited the fluid secretion induced by Danshen (263% ± 63% vs 309% ± 45%, 227% ± 63% vs 309% ± 45%, P = 0.899, 0.626 > 0.05 respectively, by ANOVA). Accordingly, Danshen does not bind with M3 or α1 receptors. These characteristics suggested that the mechanism involved in DS-induced salivary fluid secretion could be different from that induced by carbamylcholine. Carbamylcholine activates the M3 receptor to release inositol trisphosphate (IP3) and quickly releases Ca2+ from the calcium stores. The elevation of [Ca2+]i induces chloride release and quick osmosis, resulting in an onset of fluid secretion. An increase in [Ca2+]i is essential for the activation of the luminal Cl- and basolateral K+ channels. The nominal removal of extracellular Ca2+ totally abolished the fluid secretion induced by Danshen (1.8 ± 0.8 μL/g-min vs 101.9 ± 17.2 μL/g-min, P = 0.00023 < 0.01, by t-test), suggesting the involvement of Ca2+ in the activation of these channels. Therefore, IP3-store Ca2+ release signalling may not be involved in the secretion induced by Danshen, but rather, there may be a distinct signalling process.

CONCLUSION: The present findings suggest that Danshen can be used in the treatment of xerostomia, to avoid the systemic side effects associated with muscarinic drugs.

Keywords: Salivary fluid secretion; Xerostomia; Chinese herb; Danshen; Submandibular gland; Oxygen consumption; Extracellular Ca2+

Core tip: Perfusion of rat submandibular glands allowed for the measurement of fluid secretion and oxygen consumption during Danshen stimulation, which induced a large salivary fluid secretion. Ouabain (Na+/K+ ATPase inhibitor) and bumetanide (NKCC1 inhibitor), inhibited fluid secretion and oxygen consumption significantly, indicating that Danshen has a similar basic mechanism for secretion. Receptor blockers indicated that Danshen does not bind with M3 or α1 receptors. These findings suggest that the mechanism for Danshen-induced fluid secretion could be different from that induced by carbamylcholine. Accordingly, Danshen may avoid the systemic side effects caused by muscarinic drugs in the treatment of xerostomia.