P2X7 Receptor-mediated Membrane Blebbing in Salivary Epithelial Cells

High concentrations of ATP induce membrane blebbing. However, the underlying mechanism involved in epithelial cells remains unclear. In this study, we investigated the role of the P2X7 receptor (P2X7R) in membrane blebbing using Par C5 cells. We stimulated the cells with 5 mM of ATP for 1~2 hrs and found the characteristics of membrane blebbing, a hallmark of apoptotic cell death. In addition, 500 micrometer Bz-ATP, a specific P2X7R agonist, induced membrane blebbing. However, 300 micrometer of Ox-ATP, a P2X7R antagonist, inhibited ATP-induced membrane blebbing, suggesting that ATP-induced membrane blebbing is mediated by P2X7R. We found that ATP-induced membrane blebbing was mediated by ROCK I activation and MLC phosphorylation, but not by caspase-3. Five mM of ATP evoked a biphasic [Ca2+]i response; a transient [Ca2+]i peak and sustained [Ca2+]i increase secondary to ATP-stimulated Ca2+ influx. These results suggest that P2X7R plays a role in membrane blebbing of the salivary gland epithelial cells.

Ca2 -activated K Currents of Pancreatic Duct Cells in Guinea-pig

There are numerous studies on transepithelial transports in duct cells including Cl and/or HCO3. However, studies on transepithelial K transport of normal duct cells in exocrine glands are scarce. In the present study, we examined the characteristics of K currents in single duct cells isolated from guinea pig pancreas, using a whole-cell patch clamp technique. Both Cl and K conductance were found with KCl rich pipette solutions. When the bath solution was changed to low Cl, reversal potentials shifted to the negative side, 75 4 mV, suggesting that this current is dominantly selective to K. We then characterized this outward rectifying K current and examined its Ca2 dependency. The K currents were activated by intracellular Ca2. 100 nM or 500 nM Ca2 in pipette significantly (P< 0.05) increased outward currents (currents were normalized, 76.8 7.9 pA, n=4 or 107.9 35.5 pA, n=6) at 100 mV membrane potential, compared to those with 0 nM Ca2 in pipette (27.8 3.7 pA, n=6). We next examined whether this K current, recorded with 100 nM Ca2 in pipette, was inhibited by various inhibitors, including Ba2, TEA and iberiotoxin. The currents were inhibited by 40.4 % (n=3), 87.0 % (n=5) and 82.5 % (n=9) by 1 mM Ba2, 5 mM TEA and 100 nM iberiotoxin, respectively. Particularly, an almost complete inhibition of the current by 100 nM iberiotoxin further confirmed that this current was activated by intracellular Ca2. The K current may play a role in secretory process, since recycling of K is critical for the initiation and sustaining of Cl or HCO3 secretion in these cells.