Activation-Dependent Cumulative Depletions of Extracellular Free Calcium in Guinea Pig Atrium Measured with Antipyrylazo III and Tetramethylmurexide

We have used a spectrophotometric method to monitor mean free extracellular calcium concentrations in isolated left atria of guinea pigs via extracellular application of the calcium-sensitive absorption dyes, antipyrylazo III and tetramethylmurexide. Exchange of extracellular free calcium with the bathing medium takes several minutes and closely parallels contractile response in this preparation. Under conditions favoring a rapid positive force staircase response during repetitive stimulation after a long rest period (2-10 minutes), cumulative depletions of extracellular calcium can clearly be differentiated from motion artifact due to muscle movement by multiple-wavelength spectrophotometry. Responses of similar magnitude and characteristics are obtained with both dyes employed. In the presence of 10 m isoproterenol, the mean extracellular calcium concentration falls by at least 5% (0.25-0.8 mm total calcium concentration) in four beats at 0.5 Hz; extracellular calcium replenishes during rest with an apparent t½ of 25-60 seconds. A 10-minute pretreatment with 10 m ryanodine greatly reduces the contraction force and motion artifact of the first beat after a rest period, whereby the magnitude of depletion response to one post-rest stimulation is increased 2- to 3-fold. With further ryanodine treatment, the magnitude of depletion responses remains stable, and the rate of calcium replenishment during rest increases many-fold. After ryanodine treatment and 10 m isoproterenol, at least 10% of total dye accessible calcium (0.25-1.0 mm) is lost during two to five rapid stimulations, and returns to the extracellular space within 20 seconds of rest. Cumulative extracellular calcium depletion responses are strongly suppressed by 10 m nifedipine. Cumulative depletion responses are also inhibited by 10 mm caffeine, whereby contraction and corresponding motion artifacts are increased at post-rest stimulation. (Circ Res 53:779-793, 1983)