NAADP is a calcium mobilising compound acting via a two-pool mechanism, with roles for two intracellular Ca2+ stores (the lysosome and the sarcoplasmic reticulum (SR)) (1). In both atrial and ventricular myocytes, NAADP increases cellular Ca2+ transients through enhancement of SR Ca2+ load (2). Our working hypothesis is that actions of NAADP involve Ca2+ release from the lysosome via a two-pore Ca2+ channel and that this in turn leads to additional Ca2+being loaded into the SR. Tissue levels of NAADP are raised after whole-heart perfusion with isoprenaline, suggesting a role for NAADP in the beta-adrenergic response (2). In sea-urchin eggs actions of NAADP can be suppressed by either bafilomycin (which inhibits a V-type Ca2+ ATP-ase in the lysosomal membrane) (1) or by NED-19 which non-competitively inhibits the action of NAADP at two-pore Ca2+ channels (3). Further, bafilomycin superfusion has been shown to reduce ventricular myocyte Ca2+ transient amplitude (2). This project aimed to measure the response of cardiac myocytes to NAADP and investigate the relevance of this pathway to beta-adrenergic signalling. All experiments were carried out using isolated guinea pig myocytes (see (4) for atrial isolations and (2) for ventricular isolations). Cells were loaded with fluo 5F calcium indicator and stimulated to fire action potentials. All data are presented as mean ± SEM. Statistical analyses were carried out using Students t-test or ANOVA with Tukey correction as appropriate. Significance is defined as p<0.05. To investigate the cellular response to NAADP cells were loaded with a ‘caged’ compound, which is inactive until exposed to UV light. UV photorelease of NAADP elicited a significant increase in cellular Ca2+ transient amplitude in both atrial and ventricular myocytes, significant by 3min and reaching 35±13 and 42±11% respectively after 5min. This response was completely abolished when photorelease was carried out in the presence of NED-19. Application of bafilomycin to atrial myocytes reduced Ca2+ transient amplitude by 19±5% from control. In the continued presence of bafilomycin, the increase in Ca2+ transient amplitude elicited by isoprenaline was significantly reduced in both atrial and ventricular myocytes (from 63±8 to 36±9% and from 148±6 to 102±12% respectively). Further, in the presence of NED-19, the response of ventricular myocytes to isoprenaline was reduced from 148±6% to 105±6%. Isoprenaline responses in the presence of bafilomycin and NED-19 were not significantly different. These data support the two-pool model for NAADP actions in cardiac myocytes. It appears from these observations that the NAADP system is constitutively active, and has a physiological role in cardiac signalling that is modulated by beta-adrenoceptor stimulation.