Recent studies on cardiac pacemaker cells have demonstrated that interventions which affect intracellular Ca2+ concentration ([Ca2+]i) also influence firing rate (Zhou & Lipsius, 1993; Ju & Allen, 1998; Bogdanov et al., 2001). To investigate the involvement of [Ca2+]i in modulation of heart rate by ATP, we examined the effect of extracellular ATP (10-100 μM) on [Ca2+]i and spontaneous firing rate in single pacemaker cells isolated from the sinus venosus of cane toads*. In spontaneously firing cells, ATP initially increased peak [Ca2+]i, diastolic [Ca2+]i, and the firing rate. These early effects were followed by a late phase in which the peak [Ca2+]i, diastolic [Ca2+]i and the firing rate all declined. Previous studies suggested that positive phase was mediated by P2 purinoceptors, activated by ATP, while the negative phase was mediated by P1 purinoceptors, activated by adenosine (Burnstock & Meghji, 1981). To define the subtype of purinoceptors involved we used αβ-methylene ATP, adenosine, and UTP (respectively P2X1,3, P1 and P2Y2,4,6 selective agonists). However, we found that these agonists caused no significant change in [Ca2+]i and had little or no effect on firing rate. In contrast the P2Y1 selective agonist 2-MesADP (1 μM) mimicked the biphasic effects of ATP and these effects were inhibited by the non-selective purinoceptor antagonist suramin and by the P2Y1 selective antagonist MSR 2179.
Immunohistochemistry using an anti-P2Y1 antibody demonstrated that P2Y1 receptors were present on the cell surface. To establish the specificity of the antibody we performed Western blotting analysis on the protein extracts from toad tissues including sinus venosus and aorta as well as rat aorta as positive control. The immunoreaction with the P2Y1 antibody resulted in a major band of apparent molecular weight of approximately 57 kDa in all three samples. Thus the P2Y1 antibody recognized a similar molecular weight protein in both amphibian and mammalian tissues as reported by others (Moore et al., 2000).
To investigate the nature of the biphasic response we studied the effect of ATP on Ca2+ store content. We found that the effects of ATP were related to the sarcoplasmic reticulum (SR) Ca2+ store. After depletion of the SR Ca2+ store with caffeine or ryanodine, ATP no longer had any effect on [Ca2+]i or firing rate. Furthermore, the SR Ca2+ store content was decreased during the late phase of 2-MesADP application. The effect of ATP was coupled to phospholipase C (PLC) activity because the PLC inhibitor U-73122 eliminated the effect of ATP.
Our study shows that in toad pacemaker cells, the biphasic effects of ATP on pacemaker activity are mainly through P2Y1 purinoceptors, which are able to modulate Ca2+ release from the SR Ca2+ store. We propose that inositol 1,4,5-triphosphate generated by PLC facilitates SR Ca2+ release causing the early increase in peak [Ca2+]i. The increased Ca2+ release partially depletes the SR Ca2+ store accounting for the subsequent decline in peak [Ca2+]i.
Bogdanov, K.Y., Vinogradova, T.M., & Lakatta, E.G. (2001) Circulation Research 88, 1254-1258.
Burnstock, G. & Meghji, P. (1981) British Journal of Pharmacology 73, 879-885.
Ju, Y-K & Allen, D.G. (1998) Journal of Physiology 508, 153-166.
Moore, D., Chambers, J., Waldvogel, H., Faull, R., & Emson, P. (2000) Journal of Comparative Neurology 421, 347-384.
Zhou, Z. & Lipsius, S.L. (1993) Journal of Physiology 466, 263-285.