Intracellular calcium and pacemaker activity in the sinoatrial node
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The importance of the sinoatrial (S-A) node as the pacemaker of the heart is well known; however, little is known about calcium homeostasis and excitation-contraction coupling in the pacemaker cells. Thus, the major objectives of this dissertation research were: (1) to quantitatively measure intracellular calcium (Cai^2+) in isolated pacemaker cells; (2) to examine the relationship between Cai^2+ and electrical activity; and (3) to measure Ca^2+ release from the sarcoplasmic reticulum (SR) and determine its effect on pacemaker activity. We used the fluorescent Ca^2+ indicator, Indo-1, to assess the changes in Cai^2+ and perforated-patch whole-cell recordings to monitor the electrical activity of single, cultured pacemaker cells that had been isolated from the rabbit S-A node. Indo-1 fluorescence ratios were calibrated both in vitro and in vivo. The contributions to action potential-induced Cai^2+ transients from Ca^2+ entry through voltage-gated channels and Ca^2+ release from the SR were demonstrated. The results indicate the existence of an internal Ca^2+ store, the SR, and that action potential-induced Cai^2+ transients are produced mainly by Ca^2+ entry through voltage-gated Ca^2+ channels, rather than by Ca^2+ release from the SR. Simultaneous recordings of membrane potentials and the Indo-1 fluorescence ratio showed that automaticity can be modified by isoproterenol and caffeine, agents that modulate Cai^2+. Changes in beat rate were correlated with changes in Cai^2+; however, Cai^2+ transients were not essential for cells to generate spontaneous firing. Using the perforated-patch voltage-clamp technique, we studied the effects of ryanodine and thapsigargin, inhibitors of SR Ca^2+ release, on pacemaker activity, hyperpolarization-activated inward current, time-independent inward current, L- and T-type Ca ^2+ currents, and inward Na^+-Ca^2+ exchange current. Both ryanodine and thapsigargin slowed pacemaker activity significantly, in part by reducing inward Na^+-Ca^2 exchange current; ryanodine also had a direct inhibitory effect on T-type Ca^2+ current. In summary, rabbit S-A node pacemaker cells display a complex set of positive and negative feedback mechanisms that control intracellular Ca^2+ and pacemaker activity.