In humans, approximately 90% of saliva is secreted by the 3 major salivary glands: the parotid (PG), the submandibular (SMG), and the sublingual glands (SLG). Even though it is known that all 3 major salivary glands secrete saliva by a Cl(-)-dependent mechanism, salivary secretion rates differ greatly among these glands. The goal of this study was to gain insight into the properties of the ion-transporting pathways in acinar cells that might account for the differences among the major salivary glands. Pilocarpine-induced saliva was simultaneously collected in vivo from the 3 major salivary glands of mice. When normalized by gland weight, the amount of saliva secreted by the PG was more than 2-fold larger than that obtained from the SMG and SLG. At the cellular level, carbachol induced an increase in the intracellular [Ca(2+)] that was more than 2-fold larger in PG and SMG than in SLG acinar cells. Carbachol-stimulated Cl(-) efflux and the protein levels of the Ca(2+)-activated Cl(-) channel TMEM16A, the major apical Cl(-) efflux pathway in salivary acinar cells, were significantly greater in PG compared with SMG and SLG. In addition, we evaluated the transporter activity of the Na(+)-K(+)-2Cl(-) cotransporters (NKCC1) and anion exchangers (AE), the 2 primary basolateral Cl(-) uptake mechanisms in acinar cells. The SMG NKCC1 activity was about twice that of the PG and more than 12-fold greater than that of the SLG. AE activity was similar in PG and SLG, and both PG and SLG AE activity was about 2-fold larger than that of SMG. In summary, the salivation kinetics of the 3 major glands are distinct, and these differences can be explained by the unique functional properties of each gland related to Cl(-) movement, including the transporter activities of the Cl(-) uptake and efflux pathways, and intracellular Ca(2+) mobilization.
OBJECTIVE Non-obese diabetic (NOD) mice develop an autoimmune exocrinopathy that shows similarities with Sjögren’s syndrome. They provide an experimental model to study the pathoetiogenesis of this disease. MATERIALS AND METHODS Salivary gland (SG) function and salivary sodium content were measured in 8-, 12-, 16- and 20-week-old NOD and age-matched CB6 mice. In NOD mice, SG expression of phenotypic cell markers, B cell-stimulating and costimulatory molecules were evaluated. Cytokine levels were measured in serum and SG homogenates. RESULTS Microscopically evident SG inflammation in NOD mice was preceded by expression of intercellular adhesion molecule 1 on epithelial cells in the presence of macrophages and relatively high levels of cytokines. Next, an influx consisting of mainly T, B, natural killer, plasma and dendritic cells was seen. Most cytokines, except for interleukin (IL)12 /IL23p40 and B cell-activating factor, decreased or remained stable over time, while glandular function deteriorated from 16 weeks of age onward compared with CB6 mice. CONCLUSION Sjögren’s syndrome-like disease in NOD mice occurs in multiple stages; immunological and physiological abnormalities can be detected before focal inflammation appears and salivary output declines. Extrapolating this knowledge to human subjects could help in understanding the pathogenesis and aid the identification of potential therapeutic targets.
Na+:K+:2Cl− cotransporters (NKCCs) belong to the SLC12A family of cation-coupled Cl− transporters. We investigated whether enamel-producing mouse ameloblasts express NKCCs. Transcripts for Nkcc1 were identified in the mouse dental epithelium by RT-qPCR and NKCC1 protein was immunolocalized in outer enamel epithelium and in the papillary layer but not the ameloblast layer. In incisors of Nkcc1-null mice late maturation ameloblasts were disorganized, shorter and the mineral density of the enamel was reduced by 10% compared to wild-type controls. Protein levels of gap junction protein connexin 43, Na+-dependent bicarbonate cotransporter e1 (NBCe1), and the Cl−-dependent bicarbonate exchangers SLC26A3 and SLC26A6 were upregulated in Nkcc1-null enamel organs while the level of NCKX4/SLC24A4, the major K+, Na+ dependent Ca2+ transporter in maturation ameloblasts, was slightly downregulated. Whole-cell voltage clamp studies on rat ameloblast-like HAT-7 cells indicated that bumetanide increased ion-channel activity conducting outward currents. Bumetanide also reduced cell volume of HAT-7 cells. We concluded that non-ameloblast dental epithelium expresses NKCC1 to regulate cell volume in enamel organ and provide ameloblasts with Na+, K+ and Cl− ions required for the transport of mineral- and bicarbonate-ions into enamel. Absence of functional Nkcc1 likely is compensated by other types of ion channels and ion transporters. The increased amount of Cx43 in enamel organ cells in Nkcc1-null mice suggests that these cells display a higher number of gap junctions to increase intercellular communication.
The mechanisms underlying the functional differences in sympathetic and parasympathetic regulation of the major salivary glands have received little attention. The acute effects of parasympathetic muscarinic (carbachol)-dependent and combined parasympathetic-dependent plus cAMP-dependent pathways on fluid secretion rates, ion composition, and protein content were assessed using a newly developed ex vivo preparation that allows the simultaneous perfusion of the mouse submandibular (SMGs) and sublingual glands (SLGs). Our results confirm that the muscarinic-dependent pathway accounts for the bulk of salivation in SMGs and SLGs, whereas costimulation with a cAMP-increasing agent (forskolin, isoproterenol, or vasoactive intestinal peptide) did not increase the flow rate. Costimulation with carbachol plus the β-adrenergic agonist isoproterenol decreased the concentration of NaCl and produced a substantial increase in the protein and Ca2+ content of SMG but not SLG saliva, consistent with a sparse sympathetic innervation of the SLGs. On the other hand, forskolin, which bypasses receptors to increase intracellular cAMP by directly activating the enzyme adenylate cyclase, enhanced the secretion of protein and Ca2+ by both the SMGs and SLGs. In contrast, isoproterenol and vasoactive intestinal peptide specifically stimulated protein secretion in SMG and SLG salivas, respectively. In summary, cAMP-dependent signaling does not play a major role in the stimulation of fluid secretion in SMGs and SLGs, whereas each cAMP-increasing agonist behaves differently in a gland-specific manner suggesting differential expression of G protein-coupled receptors in the epithelial cells of SMGs and SLGs.
Senecio nutans Sch. Bip. and its constituents are reported to have antihypertensive effects. We isolated metabolite–1, a natural compound from S. nutans (4-hydroxy-3-(isopenten-2-yl)-acetophenone), and synthesized novel oxime – 1 (4-hydroxy-3-(isopenten-2-yl)-acetophenoxime) to evaluate their effect on vascular reactivity. Compounds were purified (metabolite–1) or synthetized (oxime–1) and characterized using IR and NMR spectroscopy and Heteronuclear Multiple Quantum Coherence (HMQC). Using pharmacological agents such as phenylephrine (PE) and KCl (enhancing contraction), acetylcholine (ACh), L-NAME (nitric oxide (NO) and endothelial function), Bay K8644-induced CaV1.2 channel (calcium channel modulator), and isolated aortic rings in an organ bath setup, the possible mechanisms of vascular action were determined. Pre-incubation of aortic rings with 10−5 M oxime–1 significantly (p < 0.001) decreased the contractile response to 30 mM KCl. EC50 to KCl significantly (p < 0.01) increased in the presence of oxime–1 (37.72 ± 2.10 mM) compared to that obtained under control conditions (22.37 ± 1.40 mM). Oxime–1 significantly reduced (p < 0.001) the contractile response to different concentrations of PE (10−7 to 10−5 M) by a mechanism that decreases Cav1.2-mediated Ca2+ influx from the extracellular space and reduces Ca2+ release from intracellular stores. At a submaximal concentration (10−5 M), oxime–1 caused a significant relaxation in rat aorta even without vascular endothelium or after pre-incubate the tissue with L-NAME. Oxime–1 decreases the contractile response to PE by blunting the release of Ca2+ from intracellular stores and blocking of Ca2+ influx by channels. Metabolite–1 reduces the contractile response to KCl, apparently by reducing the plasma membrane depolarization and Ca2+ influx from the extracellular space. These acetophenone derivates from S. nutans (metabolite–1 and oxime–1) cause vasorelaxation through pathways involving an increase of the endothelial NO generation or a higher bioavailability, further highlighting that structural modification of naturally occurring metabolites can enhance their intended pharmacological functions.
In mammals, the daily variation in the ecology of the intestinal microbiota is tightly coupled to the circadian rhythm of the host. On the other hand, a close correlation between increased body weight and light pollution at night has been reported in humans and animal models. However, the mechanisms underlying such weight gain in response to light contamination at night remain elusive. In the present study, we tested the hypothesis that dim light pollution at night alters the colonic microbiota of mice, which could correlate with weight gain in the animals. By developing an experimental protocol using a mouse model that mimics light contamination at night in urban residences (dLAN, dim light at night), we found that mice exposed to dLAN showed a significant weight gain compared with mice exposed to control standard light/dark (LD) photoperiod. To identify possible changes in the microbiota, we sampled two stages from the resting period of the circadian cycle of mice (ZT0 and ZT10) and evaluated them by high-throughput sequencing technology. Our results indicated that microbial diversity significantly differed between ZT0 and ZT10 in both LD and dLAN samples and that dLAN treatment impacted the taxonomic composition, functions, and interactions of mouse colonic microbiota. Together, these results show that bacterial taxa and microbial metabolic pathways might be involved with the mechanisms underlying weight gain in mice subjected to light contamination at night.
Two pore domain potassium channels (K2P) are strongly expressed in the nervous system (CNS), where they play a central role in excitability. These channels give rise to background K+ currents, also known as IKSO (standing-outward potassium current). We detected the expression in primary cultured cerebellar granule neurons (CGNs) of TWIK-1 (K2P1), TASK-1 (K2P3), TASK-3 (K2P9), and TRESK (K2P18) channels by immunocytochemistry and their association with lipid rafts using the specific lipids raft markers flotillin-2 and caveolin-1. At the functional level, methyl-β-cyclodextrin (MβCD, 5 mM) reduced IKSO currents by ~40% in CGN cells. To dissect out this effect, we heterologously expressed the human TWIK-1, TASK-1, TASK-3, and TRESK channels in HEK-293 cells. MβCD directly blocked TASK-1 and TASK-3 channels and the covalently concatenated heterodimer TASK-1/TASK-3 currents. Conversely, MβCD did not affect TWIK-1- and TRESK-mediated K+ currents. On the other hand, the cholesterol-depleting agent filipin III did not affect TASK-1/TASK-3 channels. Together, the results suggest that neuronal background K+ channels are associated to lipid raft environments whilst the functional activity is independent of the cholesterol membrane organization.
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