Caffeine-induced Ca²⁺ release involves four phases that require active SERCA pumps in smooth muscle cells.

<p>Single freshly isolated smooth muscle cells from guinea pig urinary bladder were loaded with both fura-2 and Mag-Fluo-4 to measure [Ca²⁺]_i and [Ca²⁺]_FSR, respectively. (A) Caffeine application (20 mM with a puffer pipette for 5 seconds) induced a transient increase of the [Ca²⁺]_i and a transient reduction of the [Ca²⁺]_FSR. Inhibition of SERCA pumps with thapsigargin reduced both the amplitude and the rate of [Ca²⁺]_i response; however, (B) close examination of these responses show that caffeine induced an increase in the [Ca²⁺]_i before any reduction of the [Ca²⁺]_FSR (phase 1) followed by a sharp reduction in the [Ca²⁺]_FSR without any effect on the [Ca²⁺]_i (phase 2). Interestingly, the inhibition of SERCA pumps with thapsigargin fused phase1 and phase 2 in a linear reduction of the [Ca²⁺]_FSR associated with a smaller increase of the [Ca²⁺]_i. Ca²⁺ recording were carried out as previously described (10).