The effects of tetracaine were studied on voltage‐clamped rat ventricular myocytes, which exhibited Ca2+ overload as identified by supontaneous Ca2+ release from the sarcoplasmic reticulum (SR) as shown by the associated contractions. This Ca2+ release was initially abolished by tetracaine before returning at a lower frequency, but greater amplitude, than the control. On removal of tetracaine, there was a burst of supontaneous Ca2+ release activity. All these effects were dose dependent, from 25 to 200 μm tetracaine. The supontaneous Ca2+ release activated an inward Na+–Ca2+ exchange current as Ca2+ was pumped out of the cell. The integral of this current (i.e. the Ca2+ efflux) was increased in the presence of tetracaine. The calcium efflux per unit time was unaffected by tetracaine. The SR Ca2+ content was increased by tetracaine, as shown by the integral of the caffeine‐evoked Na+–Ca2+ exchange current. The increase of SR Ca2+ content was equal to the extra Ca2+ lost from the cell during the burst on removal of tetracaine, and to estimates of the extra calcium gained over the quiescent period following addition of tetracaine. It is concluded that partial inhibition of calcium‐induced calcium release increases SR Ca2+ content. In the steady state, cell Ca2+ balance is maintained as the lower frequency of supontaneous release (that activates efflux) is compensated for by their greater size.

The effects of tetracaine were examined on rat ventricular myocytes. In both field‐stimulated and voltage‐clamped cells tetracaine (100–200 μM) produced an initial decrease of contraction before a recovery towards the control level. Removal of tetracaine produced a transient overshoot of contraction to levels greater than the control. The transient decrease of contraction produced by tetracaine was accompanied by a small transient increase in the integral of the L‐type Ca2+ current and a larger transient decrease of the Na+‐Ca2+ exchange current on repolarization. These are attributed to decreased systolic release of Ca2+. On removal of tetracaine there was an increase of the Na+‐Ca2+ exchange current. Before the addition of tetracaine, calculated Ca2+ influx and efflux across the sarcolemma were approximately equal. On adding tetracaine, efflux was transiently less than influx and, on removal of tetracaine, efflux was greater than influx. These changes in Ca2+ fluxes result in an increase of cell Ca2+ during exposure to tetracaine. The calculated magnitude of this increase was equal to that measured directly by applying caffeine (20 mM) to release sarcoplasmic reticulum (SR) Ca2+ and integrating the resulting Na+‐Ca2+ exchange current. It is concluded that the effects of tetracaine can be accounted for by depression of calcium‐induced Ca2+ release (CICR). The response is transient because the inhibition is compensated for by an increase of SR Ca2+ content such that there is no steady‐state effect on the magnitude of the systolic Ca2+ transient. The consequences of this result for the effects of other modulators of CICR are discussed.

Changes in the behavior of the sarcoplasmic reticulum (SR) in rat ventricular myocytes were investigated under conditions of metabolic inhibition using laser-scanning confocal microscopy to measure intracellular Ca 2 and the perforated patch-clamp technique to measure SR Ca 2 content. Metabolic inhibition had several effects on SR function, including reduced frequency of spontaneous releases of Ca 2 (sparks and waves of Ca 2-induced Ca 2 release), increased SR Ca 2 content (79.45.7 to 115.26.6 mol/L cell volume [meanSEM; P0.001]), and, after a wave of Ca 2 release, slower reuptake of Ca 2 into the SR (rate constant of fall of Ca 2 reduced from 8.51.1 s 1 in control to 5.20.4 s 1 in metabolic inhibition [P0.01]). Inhibition of L-type Ca 2 channels with Cd 2 (100 mol/L) did not reproduce the effects of metabolic inhibition on spontaneous Ca 2 sparks. These results are evidence of inhibition of both Ca 2 release and reuptake mechanisms. Reduced frequency of release could be attributable to either of these effects, but the increased SR Ca 2 content at the time of reduced frequency of spontaneous release of Ca 2 shows that the dominant effect of metabolic inhibition is to inhibit release of Ca 2 from the SR, allowing the accumulation of greater than normal amounts of Ca 2. In the context of ischemia, this extra accumulation of Ca 2 would present a risk of potentially arrhythmogenic, spontaneous release of Ca 2 on reperfusion of the tissue. (Circ Res. 2001;88:181-187.) Key Words: cardiac sarcoplasmic reticulum calcium metabolic inhibition D uring myocardial ischemia, there are profound changes in many metabolite concentrations; for example, the tissue grows progressively more acid, ATP levels fall, 1 and inorganic phosphate 2 and free magnesium rise. 3 Once blood supply is renewed, there follows a large rise of intracellular Ca 2 concentration ([Ca 2 ] i). 4,5 This may be the result of reactivation of the Na-Ca 2 exchanger 6 under conditions of elevated intracellular Na concentration, favoring influx of Ca 2 and efflux of Na. As a consequence, the myocytes and, in particular, the sarcoplasmic reticulum (SR) become overloaded with Ca 2 , a state that allows spontaneous release of Ca 2 that propagates along cells. This is of major importance , because spontaneous release of Ca 2 from the SR can lead to arrhythmias. 7 The conditions after reperfusion clearly favor loading of the SR with Ca 2 ; however, relatively little is known of the loading of the SR during ischemic conditions. Many of the metabolic changes that occur during ischemia are known to inhibit both the Ca 2 ATPase and the Ca 2 release channel (ryanodine receptor [RyR]) of cardiac SR. For example, reducing [ATP] and increasing [ADP] will decrease both the activity of the SR Ca 2 ATPase and the open probability of the RyR. 8,9 Intracellular acidification will also affect both. 9,10 Therefore, the ability of the SR to accumulate Ca 2 may be compromised during ischemia, but its ability to retain Ca 2 may be improved. Therefore, it is difficult to say whether th...

Changes of the activity of the sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) affect the amplitude of the systolic Ca(2+) transient and thence cardiac contractility. This is thought to be due to alterations of SR Ca(2+) content. Recent work on mice in which the expression of SERCA is decreased found that a large reduction of SERCA expression resulted in a proportionately much smaller decrease of SR Ca(2+) content. The aim of the current work was to investigate the quantitative nature of the dependence of both the amplitude of the systolic Ca(2+) transient and SR Ca(2+) content on SERCA activity during acute partial inhibition of SERCA. Experiments were performed on rat ventricular myocytes. Brief application of thapsigargin (1 μm) resulted in a decrease of SERCA activity as measured from the rate of decay of the systolic Ca(2+) transient. This was accompanied by a decrease in the amplitude of the systolic Ca(2+) transient which was linearly related to that of SERCA activity. However, the fractional decrease in the SR Ca(2+) content was much less than that of SERCA activity. On average SR Ca(2+) content was proportional to SERCA activity raised to the 0.38 ± 0.07 power. This shallow dependence of SR content on SERCA activity arises because Ca(2+) release is a steep function of SR Ca(2+) content. In contrast SR Ca(2+) content was increased 4.59 ± 0.40 (n = 8)-fold by decreasing ryanodine receptor opening with tetracaine (1 mm). Therefore a modest decrease of SR Ca(2+) content results in a proportionately larger fall of Ca(2+) release from the SR which can balance a larger initiating decrease of SERCA. In conclusion, the shallow dependence of SR Ca(2+) content on SERCA activity is expected for a system in which small changes of SR Ca(2+) content produce larger effects on the amplitude of the systolic Ca(2+) transient.