<ORIGINAL ARTICLE>Inhibitory effects of extracellular ATP on the Ca^<2+> mobilization evoked by muscarinic stimulation in rat parotid acinar cells

In rat parotid acinar cells, extracellular ATP reduced the increase in cytosolic Ca^ concentration ([Ca^]_i) induced by the muscarinic receptor agonist carbachol (CCh) dose-dependently. This inhibitory effect was also observed in the absence of extracellular Ca^, indicating that the Ca^ release from intracellular Ca^ stores was prevented by ATP. Formation of inositol trisphosphate induced by CCh stimulation was strongly attenuated in the presence of extracellular ATP, suggesting that the effect of ATP on the CCh-induced [Ca^]_i response was the result of a decreased phosphoinositide hydrolysis. Extracellular ATP had no effect on the CCh-induced increase in [Ca^] j in rat lacrimal acinar cells that lack the purinergic receptors. This result supports the view that the inhibitory effect of ATP on the CCh-induced [Ca^]_i response in parotid acinar cells is mediated through activation of the purinergic receptros. In rat parotid acinar cells, the purinergic receptors may play a role in modulating the activity of muscarinic receptors

Expression of hyaluronan synthase 3 in deformed human temporomandibular joint discs: in vivo and in vitro studies

The present study aimed at investigating the expression of a hyaluronan synthase (HAS) 3 in tissue samples of deformed human temporomandibular joint (TMJ) discs and cells obtained from the discs. Fifteen adult human TMJ discs (twelve diseased discs and three normal discs) were used in this study. The twelve diseased discs were obtained from twelve patients with internal derangement (ID) of TMJ. These patients all had anteriorly displaced discs and deformed discs. The tissues were immunohistochemically stained using HAS3 antibodies. In addition, the subcultured TMJ disc cells under both normal and hypoxic conditions (O2: 2%) were incubated for 3, 6, 12, and 24 h after addition of interleukin-1β (IL-1β) (1 ng/mL). Subsequently, the expression of HAS3 was examined using real-time reverse transcription-polymerase chain reaction (RT-PCR). The control group showed from negative to weak positive reactions for HAS3 on immunohistochemical staining. The discs extracted from twelve cases with ID presented from moderate to strong positive reactions for HAS3. The quantity of HAS3 mRNA was compared with a control group, and showed a 204-fold increase at 3 h, a 26-fold increase at 6 h, a 2.5-fold increase at 12 h and a 32-fold increase at 24 h under hypoxia with the addition of IL-1β. The expression of HAS3 mRNA was significantly enhanced at 3 h and 24 h. The results obtained suggest that HAS3 is related to the pathological changes of human TMJ discs affected by ID

Expression of lumican related to CD34 and VEGF in the articular disc of the human temporomandibular joint

Lumican belongs to the small leucine-rich repeat proteoglycan (SLRP) gene family and has been reported to exist in the cornea, intervertebral disc and tendon. Lumican plays a significant role in the assembly and regulation of collagen fibres. The human temporomandibular joint (TMJ) disc is made up of fibrocartilage with an extracellular matrix (ECM) composed of collagen and proteoglycans. The existence and behaviour of lumican have not been studied in the human TMJ disc. Therefore, we used immunohistochemical methods to detect lumican, CD34 and vascular endothelial growth factor (VEGF) and histochemical staining with toluidine blue in 13 human TMJ specimens (10 surgically removed and 3 obtained from autopsy). In both normal and deformed discs we observed staining with toluidine blue. We found that the area of metachromasia inside the deformed disc was uneven and expression of lumican was strong in the areas negative for metachromasia. Staining of VEGF and CD34 inside the deformed disc was seen. We confirmed the expression of lumican in the human TMJ disc and showed that a large number of fibroblast-like cells existed in the area of strong lumican expression. These new findings about the behaviour of lumican suggest that it may play a key role in the generation of a new collagen network by fibroblast-like cells

カルシウムシグナルと唾液分泌機能

Calcium ion (Ca^) plays an essential role as a second messenger in secretion of water and electrolytes in salivary glands. Many studies using fluorescent Ca^ indicators such as quin-2 and fura-2 have shown that stimulation of muscarinic, α-adrenergic, or substance P receptors cause a rapid increase in [Ca^]_i in salivary gland cells resulting from the production of inositol 1,4,5-trisphosphate (IP_3). The [ra～]_i response is attributed to Ca^ release from intracellular Ca^ store followed by Ca^ entry across the plasma membrane. The increase in [Ca^]_i is initiated in the apical pole of acinar cells and then rapidly spreads as a Ca^ wave toward the basolateral region. The apical-to-basal pattern of Ca^ signaling is probably important to activate the ion transport systems in salivary glands. In this review, I discuss mechanisms and physiological significance of the polarized Ca^ signaling in salivary gland cells

Spontaneous Ca2+ oscillations via purinergic receptors elicit transient cell swelling in rat parotid ducts

Rat parotid ductal cells were found to exhibit spontaneous Ca2+ oscillations. These oscillatory Ca2+ responses were observed during continuous perfusion with physiological salt solution at 37℃ in the absence of calcium mobilizing agonist stimulation. These Ca2+ oscillations were completely blocked by the purinergic receptor inhibitors, pyridoxal phosphate-6-azo (benzene-2,4-disulfonic acid) (PPADS) and suramin, but were not blocked by the muscarinic antagonist, atropine, nor the α-adrenergic antagonist, phentolamine. Simultaneous observation with fura-2 fluorescence and differential interference contrast (DIC) images showed that the spontaneous elevations of [Ca2+]i were well correlated with the shape changes of the ductal cells. Using a plasma membrane fluorescence probe, we found that the changes in DIC images reflected spontaneous cell swelling of ductal cells. Electron microscopic analysis after Ca2+ imaging indicated that the spontaneously oscillating duct cells contained numerous granules at the luminal side, which is characteristic of the granular duct cells. These results indicate that the spontaneous [Ca2+]i increase occurs through purinergic receptors, and activates Ca2+-dependent ion transporters and/or channels. Our findings present the possibility that spontaneous Ca2+ oscillations via purinergic receptors are involved in the regulation of the electrolyte composition of saliva in resting states

Monitoring of IP3 dynamics during Ca2+ oscillations in HSY human parotid cell line with FRET-based IP3 biosensors

Inositol 1,4,5-trisphosphate (IP3) is an intracellular messenger that elicits a wide range of spatial and temporal Ca2+ signals, and this signaling versatility is exploited to regulate diverse cellular responses. In the present study, we have developed a series of IP3 biosensors that exhibit strong pH stability and varying affinities for IP3, as well as a method for the quantitative measurement of cytosolic concentrations of IP3 ([IP3]i) in single living cells. We applied this method to elucidate IP3 dynamics during agonist-induced Ca2+ oscillations, and demonstrated cell type-dependent differences in IP3 dynamics ; a non-fluctuating rise in [IP3]i and repetitive IP3 spikes during Ca2+ oscillations in COS-7 cells and HSY-EA1 cells, respectively. The size of the IP3 spikes in HSY-EA1 cells varied from 10 to 100 nM, and the [IP3]i spike peak was preceded by a Ca2+ spike peak. These results suggest that repetitive IP3 spikes in HSY-EA1 cells are passive reflections of Ca2+ oscillations, and are unlikely to be essential for driving Ca2+ oscillations. The novel method described herein as well as the quantitative information obtained by using this method should provide a valuable and sound basis for future studies on the spatial and temporal regulations of IP3 and Ca2+

A novel Stim1-dependent, non-capacitative Ca2+ entry pathway is activated by B cell receptor stimulation and depletion of Ca2+ stores

In most non-excitable cells, the depletion of intracellular Ca2+ stores activates capacitative Ca2+ entry (CCE), which is a Ca2+-selective and La3+-sensitive entry pathway. Here, we report a novel mechanism of La3+-resistant Ca2+ entry that is synergistically regulated by B cell receptor (BCR) stimulation and Ca2+ store depletion (B-SOC). In the wildtype (WT) DT40 cells, BCR stimulation with anti-IgM antibodies induced Ca2+ release and subsequent Ca2+ entry in the presence of 0.3 μMLa3+ which blocks CCE completely. In the inositol 1,4,5-trisphosphate receptor-deficient (IP3R-KO) cells, BCR stimulation elicited neither Ca2+ release nor Ca2+ entry. However, under pretreatment of thapsigargin (ThG), BCR stimulation induced La3+-resistant Ca2+ entry into both WT and IP3R-KO cells. These results indicate that BCR stimulation and Ca2+ store depletion work in concert to activate the La3+-resistant Ca2+ entry pathway. B-SOC was inhibited by tyrosine kinase inhibitor, genistein. In addition, B-SOC was completely abolished in Stim1-deficient cells and was restored by overexpression of yellow fluorescent protein (YFP)-tagged Stim1, but was unaffected by double knockdown of Orai1/Orai2. These results demonstrate a unique non-CCE pathway, in which Ca2+ entry depends on Stim1 and tyrosine kinase activation. It is likely that similar regulation of Ca2+ entry occurs in other cell types including salivary gland cells