It has been reported that significant Ca²⁺ release was evoked by the activation of L-type Ca²⁺ current in rat superior cerebral artery smooth muscle cells (Kamishima & McCarron, 1997). Here we examined whether Ca²⁺ influx through P2X receptors also triggers Ca²⁺ release in this preparation.

Male Sprague-Dawley rats (200-300 g) were rendered unconscious by exposure to a rising concentration of CO₂ and killed by exsanguination. Single smooth muscle cells were dissociated, and membrane current and Ca²⁺ transient were simultaneously determined as previously described (Kamishima & McCarron, 1997).

Application of P2X agonists evoked membrane currents and concomitant Ca²⁺ transients in whole-cell voltage-clamped single cells. The expected increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) was calculated from the time-integrated P2X current by assuming Ca²⁺ is the only permeant ion. The measured increase in [Ca²⁺]_i was plotted as a function of expected increase in [Ca²⁺]_i, and Ca²⁺ buffering power was obtained as a reciprocal of the linear fit to this relationship. Ca²⁺ buffering power of the control cells was 4752 ± 459 (mean ± S.E.M., n = 11). In the presence of a blocker of Ca²⁺-induced Ca²⁺ release, ryanodine, Ca²⁺ buffering power was 4947 ± 845 (n = 6). In the presence of a putative activator of Ca²⁺-induced Ca²⁺ release, cyclic ADP ribose, Ca²⁺ buffering power was 3996 ± 303 (n = 5). No significant difference was detected among these values by one-way ANOVA, suggesting that Ca²⁺ influx through P2X receptors does not trigger significant Ca²⁺ release in this preparation. Comparison of Ca²⁺ buffering power for L-type Ca²⁺ channels and P2X receptors suggested that about 5 % of the P2X current, corresponding to the fractional unitary current of about 25 fA at -60 mV, is carried by Ca²⁺. To test the hypothesis that P2X response may influence the subsequent P2Y response, we induced a P2Y-mediated Ca²⁺ transient with and without depolarization. The maximum rate of Ca²⁺ increase, obtained as the steepest slope of the linear regression to the rising phase of the P2Y receptor-mediated Ca²⁺ transient, was 1526 ± 446 nM s^-1 (n = 5) without depolarization. The maximum rate of Ca²⁺ increase with depolarization was 5446 ± 1224 nM s^-1 (n = 4). Student’s unpaired t test detected a significant difference in these values (P < 0.05).

Thus, membrane depolarization caused by P2X current may influence the subsequent P2Y-mediated elevation in [Ca²⁺]_i by modulating second messenger signalling cascade.

This work was supported by the British Heart Foundation (FS/2000001).