adenosine-5--o-(3-thiotriphosphate) and fura-2-am

Purinoreceptors of the P2 family contribute strongly to signaling in the cochlea, but little is known about the effects of purinergic neurotransmission in the central auditory system. Here we examine P2 receptor-mediated signaling in the large spherical bushy cells (SBCs) of Mongolian gerbils around the onset of acoustically evoked signal processing (P9-P14). Brief adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) application evoked inward current, membrane depolarization, and somatic Ca2+ signals. Moreover, ATPgammaS changed the SBCs firing pattern from phasic to tonic, when the application was synchronized with depolarizing current injection. This bursting discharge activity was dependent on [Ca2+]i and Ca2+-dependent protein kinase (PKC) activity and is presumably caused by modulation of low-threshold K+ conductance. Activation of P2Y1 receptors could not evoke these changes per se, thus it was concluded that the involvement of P2X receptors seems to be necessary. Ca2+ imaging data showed that both P2X and P2Y1 receptors mediate Ca2+ signals in SBCs where P2Y1 receptors most likely activate the PLC-IP3 (inositol trisphosphate) pathway and release Ca2+ from internal stores. Immunohistochemical staining confirmed the expression of P2X2 and P2Y1 receptor proteins in SBCs, providing additional evidence for the involvement of both receptors in signal transduction in these neurons. Purinergic signaling might modulate excitability of SBCs and thereby contribute to regulation of synaptic strength. Functionally, the increase in firing rate mediated by P2 receptors could reduce temporal precision of the postsynaptic firing, e.g., phase locking, which has an immediate effect on signal processing related to sound localization. This might provide a mechanism for adaptation to the ambient acoustic environment.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Animals, Newborn; Biophysics; Calcium; Cochlear Nucleus; Dose-Response Relationship, Drug; Electric Stimulation; Enzyme Inhibitors; Fluorometry; Fura-2; Gene Expression Regulation; Gerbillinae; In Vitro Techniques; Lysine; Membrane Potentials; Neurons; Patch-Clamp Techniques; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Receptors, Purinergic P2; Signal Transduction; Thionucleotides; Vesicular Glutamate Transport Protein 1

Whole-cell patch-clamp recordings and single-cell Ca2+ measurements were used to study the control of Ca2+ entry through the Ca2+ release-activated Ca2+ influx pathway (ICRAC) in rat basophilic leukemia cells. When intracellular inositol 1,4,5-trisphosphate (InsP3)-sensitive stores were depleted by dialyzing cells with high concentrations of InsP3, ICRAC inactivated only slightly in the absence of ATP. Inclusion of ATP accelerated inactivation 2-fold. The inactivation was increased further by the ATP analogue adenosine 5'-[gamma-thio]triphosphate, which is readily used by protein kinases, but not by 5'-adenylyl imidodiphosphate, another ATP analogue that is not used by kinases. Neither cyclic nucleotides nor inhibition of calmodulin or tyrosine kinase prevented the inactivation. Staurosporine and bisindolylmaleimide, protein kinase C inhibitors, reduced inactivation of ICRAC, whereas phorbol ester accelerated inactivation of the current. These results demonstrate that a protein kinase-mediated phosphorylation, probably through protein kinase C, inactivates ICRAC. Activation of the adenosine receptor (A3 type) in RBL cells did not evoke much Ca2+ influx or systematic activation of ICRAC. After protein kinase C was blocked, however, large ICRAC was observed in all cells and this was accompanied by large Ca2+ influx. The ability of a receptor to evoke Ca2+ entry is determined, at least in part, by protein kinase C. Antigen stimulation, which triggers secretion through a process that requires Ca2+ influx, activated ICRAC. The regulation of ICRAC by protein kinase will therefore have important consequences on cell functioning.

Topics: Adenosine; Adenosine Triphosphate; Adenosine-5'-(N-ethylcarboxamide); Alkaloids; Animals; Calcium; Calcium Channels; Cell Line; Dialysis; Egtazic Acid; Fura-2; Homeostasis; Indoles; Inositol 1,4,5-Trisphosphate; Kinetics; Leukemia, Basophilic, Acute; Maleimides; Patch-Clamp Techniques; Protein Kinase C; Protein Kinases; Rats; Receptors, Purinergic P1; Staurosporine; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured