SIMULTANEOUS MEASUREMENT OF CA2-MUSCLE CELLS IN RESPONSE TO CAFFEINE - A NOVEL-APPROACH FOR CALCULATING THE FRACTION OF CURRENT CARRIED BY CALCIUM( RELEASE AND INFLUX INTO SMOOTH)

Activation of ryanodine receptors on the sarcoplasmic reticulum of sin gle smooth muscle cells from the stomach muscularis of Bufo marinus by caffeine is accompanied by a rise in cytoplasmic [Ca2+] ([Ca2+](i)), and the opening of nonselective cationic plasma membrane channels. To understand how each of these pathways contributes to the rise in [Ca2](i), one needs to separately monitor Ca2+ entry through them. Such in formation was obtained from simultaneous measurements of ionic current s and [Ca2+](i) by the development of a novel and general method to as sess the fraction of current induced by an agonist that is carried by Ca2+. Application of this method to the currents induced in these smoo th muscle cells by caffeine revealed that similar to 20% of the curren t passing through the membrane channels activated following caffeine a pplication is carried by Ca2+. Based on this information we found that while Ca2+ entry; through these channels rises slowly, release of Ca2 + from stores, while starting at the same time, is much faster and bri efer. Detailed quantitative analysis of the Ca2+ release from stores s uggests that it most likely decays due to depletion of Ca2+ in those s tores. When caffeine was applied twice to a cell with only a brief (30 s) interval in between, the amount of Ca2+ released from stores was m arkedly diminished following the second caffeine application whereas t he current carried in pat by Ca2+ entry across the plasma membrane was not significantly affected. These and other studies described in the preceding paper indicate that activation of the nonselective cation pl asma membrane channels in response to caffeine was not caused as a con sequence of emptying of internal Ca2+ stores. Rather, it is proposed t hat caffeine activates these membrane channels either by direct intera ction or alternatively by a linkage between