<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

Activation at a distance：new paradigm of β-arrestin dependent pathways drive mitogen-activated protein kinase signaling

departmental bulletin pape

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+

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