sq-23377 and pyridoxal-phosphate-6-azophenyl-2--4--disulfonic-acid

Activation of P2X receptors by a Ca(2+)- and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein-dependent release of ATP was measured using patch-clamp recordings from dissociated guinea pig stellate neurons. Asynchronous transient inward currents (ASTICs) were activated by depolarization or treatment with the Ca(2+) ionophore ionomycin (1.5 and 3 microM). During superfusion with a HEPES-buffered salt solution containing 2.5 mM Ca(2+), depolarizing voltage steps (-60 to 0 mV, 500 ms) evoked ASTICs on the decaying phase of a larger, transient inward current. Equimolar substitution of Ba(2+) for Ca(2+) augmented the postdepolarization frequency of ASTICs, while eliminating the larger transient current. Perfusion with an ionomycin-containing solution elicited a sustained activation of ASTICs, allowing quantitative analysis over a range of holding potentials. Under these conditions, increasing extracellular [Ca(2+)] to 5 mM increased ASTIC frequency, whereas no events were observed following replacement of Ca(2+) with Mg(2+), demonstrating a Ca(2+) requirement. ASTICs were Na(+) dependent, inwardly rectifying, and reversed near 0 mV. Treatment with the nonselective purinergic receptor antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (10 microM) blocked all events under both conditions, whereas the ganglionic nicotinic antagonist hexamethonium (100 microM and 1 mM) had no effect. PPADS also blocked the macroscopic inward current evoked by exogenously applied ATP (300 microM). The presence of botulinum neurotoxin E (BoNT/E) in the whole-cell recording electrode significantly attenuated the ionomycin-induced ASTIC activity, whereas phorbol ester treatment potentiated this activity. These results suggest that ASTICs are mediated by vesicular release of ATP and activation of P2X receptors.

Topics: Adenosine Triphosphate; Animals; Barium; Botulinum Toxins; Chlorides; Exocytosis; Guinea Pigs; Hexamethonium; Ion Channels; Ionomycin; Neurons; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Receptors, Purinergic P2; Receptors, Purinergic P2X; Sodium; Stellate Ganglion; Tetradecanoylphorbol Acetate

Guanosine 5' triphosphate (GTP), acting synergistically with the nerve growth factor (NGF), enhances the proportion of neurite-bearing cells in cultures of PC12 rat pheochromocytoma cells. We studied the transduction mechanisms activated by GTP in PC12 cells and found that addition of GTP (100 microM) increased intracellular calcium concentration ([Ca(2+)](i)) in cells that were between 60 and 70% confluent. Addition of GTP also enhanced activation of NGF-induced extracellular regulated kinases (ERKs) and induced Ca(2+) mobilization. This mobilization, due to the activation of voltage-sensitive and ryanodine-sensitive calcium channels, as well as pertussis toxin-sensitive purinoceptors, modulates Ca(2+)-activated K(+) channels not involved in activation of ERKs. The results presented here indicate that GTP-triggered [Ca(2+)](i) increase may be a key event in GTP signal transduction, which can modulate activity of ERKs. The physiological importance of the GTP effect lies in its capacity to interact with the NGF-activated pathway to enhance neurite outgrowth from PC12 cells.

Topics: Animals; Barbiturates; Blotting, Western; Calcium; Calcium Channel Blockers; Cell Count; Cell Differentiation; Chelating Agents; Clotrimazole; Diagnostic Imaging; Dose-Response Relationship, Drug; Drug Synergism; Egtazic Acid; Enzyme Inhibitors; Extracellular Space; Fluorescent Antibody Technique; Fluorescent Dyes; Gallic Acid; Growth Inhibitors; Guanosine Triphosphate; Ionomycin; Ionophores; Isoxazoles; Membrane Potentials; Microscopy, Confocal; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nerve Growth Factor; Neurites; Nifedipine; PC12 Cells; Pertussis Toxin; Pyridoxal Phosphate; Rats; Signal Transduction; Suramin; Time Factors; Triazines

Volume-sensitive Cl- channels [ICl(vol)] were studied using taurine efflux and patch-clamp experiments in 9HTEo- human tracheal cells. Cells were stimulated with the Ca(2+)- elevating agents ATP and ionomycin in isotonic medium or in hypotonic solutions. ATP (100 microM) or ionomycin (1 microM) and hypotonic shock produced a synergic effect. Indeed, the resulting taurine efflux was much higher than the sum of the single effects elicited by ATP, ionomycin, or hypotonic medium. The taurine release elicited by hypotonic shock and the potentiation by ATP and ionomycin were markedly inhibited by using a Ca(2+)-free extracellular medium and by incubating the cells with the membrane-permeable 1,2-bis(2-amino- phenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester chelating agent. Patch-clamp experiments confirmed the role of Ca2+ on ICl(vol) channels. Swelling-induced taurine efflux was inhibited by reactive blue 2, suramin, and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid. Patch-clamp experiments demonstrated that these compounds shift the voltage-dependent inactivation of ICl(vol) channels toward more negative values. This study indicates that the sensitivity of ICl(vol) to cell volume changes is modulated by intracellular Ca2+ and that purinergic receptor antagonists represent a new class of CI- channel blockers.

Topics: Adenosine Triphosphate; Antigens, Viral, Tumor; Calcium; Cell Line, Transformed; Chelating Agents; Chloride Channels; Egtazic Acid; Enzyme Inhibitors; Epithelium; Humans; Hypotonic Solutions; Ionomycin; Kinetics; Membrane Potentials; Patch-Clamp Techniques; Pyridoxal Phosphate; Simian virus 40; Suramin; Taurine; Trachea; Triazines