Mechanism involved in Danshen-induced fluid secretion in salivary glands

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.