The effects of extracellular nucleotides on [Ca2+]i signalling in a human-derived renal proximal tubular cell line (HKC-8).

HKC-8 cells are a human-derived renal proximal tubular cell line and provide a useful model system for the study of human renal cell function. In this study, we aimed to determine [Ca(2+)](i) signalling mediated by P2 receptor in HKC-8. Fura-2 and a ratio imaging method were employed to measure [Ca(2+)](i) in HKC-8 cells. Our results showed that activation of P2Y receptors by ATP induced a rise in [Ca(2+)](i) that was dependent on an intracellular source of Ca(2+), while prolonged activation of P2Y receptors induced a rise in [Ca(2+)](i) that was dependent on intra- and extracellular sources of Ca(2+). Pharmacological and molecular data in this study suggests that TRPC4 channels mediate Ca(2+) entry in coupling to activation of P2Y in HKC-8 cells. U73221, an inhibitor of PI-PLC, did not inhibit the initial ATP-induced response; whereas D609, an inhibitor of PC-PLC, caused a significant decrease in the initial ATP-induced response, suggesting that P2Y receptors are coupled to PC-PLC. Although P2X were present in HKC-8, The P2X agonist, alpha,beta me-ATP, failed to cause a rise in [Ca(2+)](i). However, PPADS at a concentration of 100 microM inhibits the ATP-induced rise in [Ca(2+)](i). Our results indicate the presence of functional P2Y receptors in HKC-8 cells. ATP-induced [Ca(2+)](i) elevation via P2Y is tightly associated with PC-PLC and TRP channel.

Inositol (1,4,5)-trisphosphate receptor links to filamentous actin are important for generating local Ca2+ signals in pancreatic acinar cells.

We explored a potential structural and functional link between filamentous actin (F-actin) and inositol (1,4,5)-trisphosphate receptors (IP(3)Rs) in mouse pancreatic acinar cells. Using immunocytochemistry, F-actin and type 2 and 3 IP(3)Rs (IP(3)R2 and IP(3)R3) were identified in a cellular compartment immediately beneath the apical plasma membrane. In an effort to demonstrate that IP(3)R distribution is dependent on an intact F-actin network in the apical subplasmalemmal region, cells were treated with the actin-depolymerising agent latrunculin B. Immunocytochemistry indicated that latrunculin B treatment reduced F-actin in the basolateral subplasmalemmal compartment, and reduced and fractured F-actin in the apical subplasmalemmal compartment. This latrunculin-B-induced loss of F-actin in the apical region coincided with a reduction in IP(3)R2 and IP(3)R3, with the remaining IP(3)Rs localized with the remaining F-actin. Experiments using western blot analysis showed that IP(3)R3s are resistant to extraction by detergents, which indicates a potential interaction with the cytoskeleton. Latrunculin B treatment in whole-cell patch-clamped cells inhibited Ca(2+)-dependent Cl(-) current spikes evoked by inositol (2,4,5)-trisphosphate; this is due to an inhibition of the underlying local Ca(2+) signal. Based on these findings, we suggest that IP(3)Rs form links with F-actin in the apical domain and that these links are essential for the generation of local Ca(2+) spikes.

Lysine-fixable dye tracing of exocytosis shows F-actin coating is a step that follows granule fusion in pancreatic acinar cells.

Exocytosis, the fusion of a vesicle with the plasma membrane, involves a complex cascade of cellular events. We set out to develop a method to identify changes in the distribution of proteins associated with exocytotic events in secretory epithelial cells. Our model system, the mouse pancreatic acinar cell, contains many hundreds of secretory vesicles (zymogen granules). Cell stimulation with low, physiological concentrations of agonist leads to the exocytosis of only a few granules. Once the preparation is fixed it is impossible with light microscopy to determine which of the granules have fused. In our method we use a derivative of a fluorescent dye, lysine-fixable Texas Red dextran dye, where the dye has been conjugated to dextran containing lysine residues. This dye enters the zymogen granule on the opening of the fusion pore at the time of exocytosis. This dye can then be fixed with paraformaldehyde and the preparation used for conventional immunocytochemistry methods. In demonstrating the utility of the method we carried out experiments where we fixed the cells and counterstained F-actin with phalloidin. The results show that F-actin coating is associated specifically with dye-filled granules and therefore is a step that follows exocytosis.

Modulation of IP(3)-sensitive Ca(2+) release by 2,3-butanedione monoxime.

We describe the actions of 2,3-butanedione monoxime (BDM) on calcium responses in secretory cells. Our studies were prompted by the widespread use of BDM as a myosin-ATPase inhibitor. Application of 10 mM BDM almost completely inhibited agonist-evoked amylase secretion from mouse pancreatic acinar cells. This action might be interpreted as indicating a role for myosin in secretion. However, BDM alone elicited a calcium response in single cells and this calcium signal was sufficient to activate calcium-dependent chloride currents. Furthermore, in some cases, BDM potentiated agonist-evoked calcium signals but almost always blocked agonist-evoked calcium oscillations. These effects of BDM were not due to an action on calcium influx pathways but rather to direct effects on IP(3)-sensitive stores. We conclude that BDM cannot be used for unequivocal identification of the involvement of myosin motors in a cellular response. Further, our evidence suggests that BDM can act directly to modify the opening of IP(3) receptors.