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

Activation of ryanodine receptors on the sarcoplasmic reticulum of single 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 information was obtained from simultaneous measurements of ionic currents and [Ca2+](i) by the development of a novel and general method to assess the fraction of current induced by an agonist that is carried by Ca2+. Application of this method to the currents induced in these smooth muscle cells by caffeine revealed that similar to 20% of the current passing through the membrane channels activated following caffeine application 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 briefer. Detailed quantitative analysis of the Ca2+ release from stores suggests that it most likely decays due to depletion of Ca2+ in those stores. 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 markedly diminished following the second caffeine application whereas the 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 plasma membrane channels in response to caffeine was not caused as a consequence of emptying of internal Ca2+ stores. Rather, it is proposed that caffeine activates these membrane channels either by direct interaction or alternatively by a linkage between