Distinct Ca2+ signalling mechanisms induced by ATP and sphingosylphosphorylcholine in porcine aortic smooth muscle cells

1 The increase in the cytosolic Ca2+ concentration ([Ca2+](i)) following re petitive stimulation with ATP or sphingosylphosphorylcholine (SPC) in singl e porcine aortic smooth muscle cells was investigated using the Ca2+ indica tor, fura-2. 2 The ATP-induced [Ca2+](i) increase resulted from both Ca2+ release and Ca 2+ influx. The former was stimulated by phospholipase C activation, while t he latter occurred predominantly via the receptor-operated Ca2+ channels (R OC), rather than the store-operated Ca2+ channels (SOC) or the voltage-oper ated Ca2+ channel (VOC). Furthermore, the P2X(5) receptor was shown to be r esponsible for the ATP-induced Ca2+ influx. 3 A reproducible [Ca2+](i) increase was induced by repetitive ATP stimulati on, but was abolished by removal of extracellular Ca2+ or inhibition of int racellular Ca2+ release using U-73122 or thapsigargin, and was restored by Ca2+ readdition in the former case. 4 SPC only caused Ca2+ release, and the amplitude of the repetitive SPC-ind uced [Ca2+](i) increases declined gradually. However, a reproducible [Ca2+] (i) increase was seen in cells in which protein kinase C being inhibited, w hich increased the SPC-induced Ca2+ influx, rather than IP3 generation. 5 In conclusion, although the amplitude of the ATP-induced Ca2+ release, me asured when Ca2+ influx was blocked, or of the Ca2+ influx when Ca2+ releas e was blocked, progressively decreased following repetitive stimulation, th e overall [Ca2+](i) increase for each stimulation under physiological condi tions remained the same, suggesting that the Ca2+ stores were replenished b y an influx of Ca2+ during stimulation. The SPC-induced [Ca2+](i) increase resulted solely from Ca2+ release and decreased gradually following repetit ive stimulation, but the decrease could be prevented by stimulating Ca2+ in flux, further supporting involvement of the intracellular Ca2+ stores in Ca 2+ signalling.