The sinoatrial node governed the heart rhythm and contraction due to constitutively active, Ca2+-activated adenylyl cyclase (AC) that generates a high level of cAMP/protein kinase A (PKA) dependent which localized submembrane Ca2+ cycling. Our earlier studies have shown that this rhythmic Ca2+ cycling ignites the SANC surface membrane. This rhythmic mechanism particularly Ca2+ cycling and cell contraction utilize ATP. To explore how ATP supply matches the demand in SANC we measured cellular ATP levels in the following group: control, during PKA inhibition by specific inhibitor peptide 15 microM PKI or by 6 microM H-89;during intercellular Ca2+ buffering by 25 microM BAPTA-AM;during activation of muscarinic receptors by carbacol 1microM+/- muscarinic inhibition by atropine 10 microM. ATP levels were compared as the change from control normalized to the extent to which 2,4- dinitrophenol reduced ATP levels after 15 min of incubation in each experiments. Inhibiting PKA by PKI or H-89, which each blocked the three major ATP consumption process (spontaneous APs, Ca2+ transients and cell contraction) depleted ATP by -44.8+/-6.2% and -43.51+/-4.9%, respectively. Thus, in spite of stooping major ATP consumption process in SANs (i.e, Ca2+-PKA and contraction) we found that cellular ATP levels significantly drop, suggesting that these inhibitors disproportionately reduce ATP production relative to consumption. Buffering the intercellular Ca2+ depleted ATP by -54+/-7.8%. Hence, the intercellular Ca2+ has not only direct effect on the surface membrane potential, but also on regulating ATP production. Such control mechanisms are apparently utilized by nature, because, similar to artificially blocking these intrinsic pathways, stimulating muscarinic receptors depleted the ATP by 45+/-10% in blocking the three major ATP consumption process. Notably, atropine was able to partially reverse the carbachol effect, the combination resulting in only 18+/-3% ATP depletion. Conversely, carbacol has minor effect on ATP level in rabbit ventricular cells. Appling charbacol to stimulated (3 Hz) and quiescent cells depleted ATP by 5.8+/-3% and 0.4+/-1.4%, respectively. These data suggest that the same signals that drive, and derive from, the utilization of ATP (i.e., by Ca2+ cycling and contractile processes) also tightly couple the production of ATP to match energy demand. We propose that the reduction in ATP in SANCs after muscarinic activation might serve as a fail-safe mechanism to slow the heart beat. The nature of these signals and the ultimate effects of ATP reduction in SANC deem further study.
