sk&f-96356 and 2-aminoethoxydiphenyl-borate

Since little is known about the role of P2Y receptors (purinoceptors) in duodenal mucosal bicarbonate secretion (DMBS), we sought to investigate the expression and function of these receptors in duodenal epithelium. Expression of P2Y(2) receptors was detected by RT-PCR in mouse duodenal epithelium and SCBN cells, a duodenal epithelial cell line. UTP, a P2Y(2)-receptor agonist, but not ADP (10 microM), significantly induced murine duodenal short-circuit current and DMBS in vitro; these responses were abolished by suramin (300 microM), a P2Y-receptor antagonist, or 2-aminoethoxydiphenyl borate (2-APB; 100 microM), a store-operated channel blocker. Mucosal or serosal addition of UTP induced a comparable DMBS in wild-type mice, but markedly impaired response occurred in P2Y(2) knockout mice. Acid-stimulated DMBS in vivo was significantly inhibited by suramin (1 mM) or PPADS (30 microM). Both ATP and UTP, but not ADP (1 microM), raised cytoplasmic-free Ca(2+) concentrations ([Ca(2+)](cyt)) with similar potencies in SCBN cells. ATP-induced [Ca(2+)](cyt) was attenuated by U-73122 (10 microM), La(3+) (30 microM), or 2-APB (10 microM), but was not significantly affected by nifedipine (10 microM). UTP (1 microM) induced a [Ca(2+)](cyt) transient in Ca(2+)-free solutions, and restoration of external Ca(2+) (2 mM) raised [Ca(2+)](cyt) due to capacitative Ca(2+) entry. La(3+) (30 microM), SK&F96365 (30 microM), and 2-APB (10 microM) inhibited UTP-induced Ca(2+) entry by 92, 87, and 94%, respectively. Taken together, our results imply that activation of P2Y(2) receptors enhances DMBS via elevation of [Ca(2+)](cyt) that likely results from an initial increase in intracellular Ca(2+) release followed by extracellular Ca(2+) entry via store-operated channel.

Topics: Adenosine Diphosphate; Aminoquinolines; Animals; Bicarbonates; Boron Compounds; Calcium Channel Blockers; Calcium Signaling; Cell Line; Dogs; Duodenum; Epithelial Cells; Estrenes; Female; Hydrogen-Ion Concentration; Intestinal Mucosa; Intestinal Secretions; Membrane Potentials; Mice; Mice, Inbred C57BL; Mice, Knockout; Nifedipine; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Pyrrolidinones; Receptors, Purinergic P2; Receptors, Purinergic P2Y2; Suramin; Time Factors; Uridine Triphosphate

Periods of low frequency stimulation are known to increase the net Ca(2+) uptake in skeletal muscle but the mechanism responsible for this Ca(2+) entry is not known. In this study a novel high-resolution fluorescence microscopy approach allowed the detection of an action potential-induced Ca(2+) flux across the tubular (t-) system of rat extensor digitorum longus muscle fibres that appears to be responsible for the net uptake of Ca(2+) in working muscle. Action potentials were triggered in the t-system of mechanically skinned fibres from rat by brief field stimulation and t-system [Ca(2+)] ([Ca(2+)](t-sys)) and cytoplasmic [Ca(2+)] ([Ca(2+)](cyto)) were simultaneously resolved on a confocal microscope. When initial [Ca(2+)](t-sys) was > or = 0.2 mM a Ca(2+) flux from t-system to the cytoplasm was observed following a single action potential. The action potential-induced Ca(2+) flux and associated t-system Ca(2+) permeability decayed exponentially and displayed inactivation characteristics such that further Ca(2+) entry across the t-system could not be observed after 2-3 action potentials at 10 Hz stimulation rate. When [Ca(2+)](t-sys) was closer to 0.1 mM, a transient rise in [Ca(2+)](t-sys) was observed almost concurrently with the increase in [Ca(2+)](cyto) following the action potential. The change in direction of Ca(2+) flux was consistent with changes in the direction of the driving force for Ca(2+). This is the first demonstration of a rapid t-system Ca(2+) flux associated with a single action potential in mammalian skeletal muscle. The properties of this channel are inconsistent with a flux through the L-type Ca(2+) channel suggesting that an as yet unidentified t-system protein is conducting this current. This action potential-activated Ca(2+) flux provides an explanation for the previously described Ca(2+) entry and accumulation observed with prolonged, intermittent muscle activity.

Topics: Action Potentials; Algorithms; Aminoquinolines; Animals; Boron Compounds; Calcium Channel Blockers; Calcium Signaling; Cell Membrane; Cytoplasm; Electric Stimulation; Fluorescent Dyes; Heterocyclic Compounds, 3-Ring; Image Processing, Computer-Assisted; Indoles; Kinetics; Microscopy, Confocal; Muscle Fibers, Skeletal; Permeability; Rats; Rats, Sprague-Dawley