INTRACELLULAR CALCIUM TRANSIENTS RECORDED WITH FURA-2 IN SPONTANEOUSLY ACTIVE MYOCYTES ISOLATED FROM THE ATRIOVENTRICULAR NODE OF THE RABBIT HEART

We have used the fluorescent Ca indicator Fura-2 to assess the changes in intracellular calcium (Ca-i) in single spontaneously active myocytes isolated from the rabbit atrioventricular node (AVN). Simultaneous recordings of membrane potential and the Fura-2 ratio signal (which reflects Ca-i) showed that a transient rise of Ca-i occurred with each spontaneous action potential (AP). The AP upstroke preceded the rise in Ca-i and repolarization of the AP occurred faster than the decline of Ca-i. The level of Ca-i remained raised and progressively declined towards a baseline diastolic level during the subsequent pacemaker depolarization. The Fura-2 (Ca-i) transient in spontaneously active AVN cells had a time-to-peak of 49.2+/-5.4ms (mean+/-s.e.m; n = 7) and declined with a single exponential time course (time constant = 139.8+/-23.9 ms; n = 7). Application of 10 mu M ryanodine completely and irreversibly abolished the Ca-i transient, identifying the sarcoplasmic reticulum (SR) as the major source of releasable Ca. Both removal of external Ca and block of L-type Ca channels (with 2 mu M nifedipine) also abolished Ca-i transients, suggesting that Ca entry via L-type Ca-channels is involved in triggering the SR Ca release underlying the Ca-i transient. Removal of external Na (in the presence of 20 mu M nifedipine to block L-type Ca channels) caused a reversible increase in Ca-i, showing that Na/Ca exchange is present in AVN cells and that it is involved in Ca-i regulation. Spontaneous Ca-i transients were abolished by 1 mu M acetylcholine, and this was associated with a hyperpolarization of membrane potential and cessation of action potentials. Isoprenaline (1 mu M) increased the rate and amplitude of spontaneous Ca-i transients; this corresponded to an increase in the rate and a change in the shape of spontaneous action potentials observed in patch-clamped AVN myocytes.