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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(-7) 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 t1/2 of 25-60 seconds. A 10-minute pretreatment with 10(-8) 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(-7) 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(-6) M nifedipine. Cumulative depletion responses are also inhibited by 10 mM caffeine, whereby contraction and corresponding motion artifacts are increased at post-rest stimulation.

Activation-dependent cumulative depletions of extracellular free calcium in guinea pig atrium measured with antipyrylazo III and tetramethylmurexide.