sq-23377 and alpha-beta-methyleneadenosine-5--triphosphate

1. The increase in the cytosolic Ca(2+) concentration ([Ca(2+)](i)) following repetitive stimulation with ATP or sphingosylphosphorylcholine (SPC) in single porcine aortic smooth muscle cells was investigated using the Ca(2+) indicator, fura-2. 2. The ATP-induced [Ca(2+)](i) increase resulted from both Ca(2+) release and Ca(2+) influx. The former was stimulated by phospholipase C activation, while the latter occurred predominantly via the receptor-operated Ca(2+) channels (ROC), rather than the store-operated Ca(2+) channels (SOC) or the voltage-operated Ca(2+) channel (VOC). Furthermore, the P2X(5) receptor was shown to be responsible for the ATP-induced Ca(2+) influx. 3. A reproducible [Ca(2+)](i) increase was induced by repetitive ATP stimulation, but was abolished by removal of extracellular Ca(2+) or inhibition of intracellular Ca(2+) release using U-73122 or thapsigargin, and was restored by Ca(2+) readdition in the former case. 4. SPC only caused Ca(2+) release, and the amplitude of the repetitive SPC-induced [Ca(2+)](i) increases declined gradually. However, a reproducible [Ca(2+)](i) increase was seen in cells in which protein kinase C being inhibited, which increased the SPC-induced Ca(2+) influx, rather than IP(3) generation. 5. In conclusion, although the amplitude of the ATP-induced Ca(2+) release, measured when Ca(2+) influx was blocked, or of the Ca(2+) influx when Ca(2+) release was blocked, progressively decreased following repetitive stimulation, the overall [Ca(2+)](i) increase for each stimulation under physiological conditions remained the same, suggesting that the Ca(2+) stores were replenished by an influx of Ca(2+) during stimulation. The SPC-induced [Ca(2+)](i) increase resulted solely from Ca(2+) release and decreased gradually following repetitive stimulation, but the decrease could be prevented by stimulating Ca(2+) influx, further supporting involvement of the intracellular Ca(2+) stores in Ca(2+) signalling.

Topics: Adenosine; Adenosine Triphosphate; Animals; Aorta; Calcium; Calcium Channel Blockers; Calcium Signaling; Cells, Cultured; Cyclic AMP; Egtazic Acid; Estrenes; Imidazoles; Ionomycin; Manganese; Muscle, Smooth, Vascular; Phosphorylcholine; Pyrrolidinones; Sphingosine; Staurosporine; Swine; Thapsigargin; Thionucleosides; Thionucleotides; Virulence Factors, Bordetella

The regulation of capacitative Ca2+ influx in Xenopus oocytes was investigated using both the two electrode voltage-clamp (where Ca2+ is monitored through the Ca2+-dependent Cl- current) and patch-clamp techniques. Following stimulation of expressed 5-hydroxytryptamine (5-HT) receptors, capacitative Ca2+ influx deactivated in around 15 min. Following injection of [adenosine 5'-O-(3-Thiotriphosphate)] (ATP [gamma-S]), an ATP analogue that is readily used by protein kinases, capacitative Ca2+ influx activated by 5-HT application either did not deactivate or was prolonged around twofold. However, injection of adenylyl 5'-(beta,gamma-methylene)-diphosphonate (AMP-PCP), another ATP analogue that is not utilised by kinases, did not affect the time-course of Ca2+ influx. When capacitative Ca2+ influx was activated by readmission of Ca2+ to oocytes incubated in thapsigargin/0 Ca2+ solution for several hours, Ca2+ influx occurred and a weakly saturating relationship between external Ca2+ and Ca2+ influx was found. Ca2+ influx in thapsigargin-treated cells was unaffected by ATP [gamma-S]. ATP [gamma-s] and several kinases had no effect on the Ca2+-dependent Cl- current when the latter was activated by elevation of Ca2+ independent of capacitative Ca2+ influx. Protein kinase C slowly and partially inhibited the Cl- current. Outside-out patches taken from thapsigargin-treated cells failed to demonstrated any Ca2+ current or Ca2+-dependent Cl- current on reapplying high Ca2+ to the patch, despite the oocyte showing a large capacitative Ca2+ influx. The results suggest that a kinase, activated on receptor stimulation, prolongs the activation time-course of capacitative Ca2+ influx.

Topics: Adenosine Triphosphate; Animals; Calcium; Calcium Channel Blockers; Chlorides; Electric Conductivity; Female; Heparin; Ionomycin; Oocytes; Patch-Clamp Techniques; Phosphoproteins; Receptors, Serotonin; Time Factors; Xenopus laevis