FIGURE 3. Caffeine does not inhibit ATP-induced increases in [Ca²⁺]_i in cultured human pulmonary artery myocytes that lack functional expression of ryanodine receptors. (A) Original recording shows that application of caffeine (10 mM) could not induce an increase in [Ca²⁺]_i in a cultured human PASMC. In the same cell, however, application of ATP (100 礛) evoked a large response in the continued presence of caffeine. (B) RyR1, RyR2, and RyR3 mRNAs are detected in cultured human PASMCs by RT-PCR. Total RNAs were amplified with oligonucleotide primers for human RyR1, RyR2, and RyR3 mRNAs, as described in MATERIALS AND METHODS. The PCR products were separated by gel electrophoresis, stained with ethidium bromide, and visualized by UV illumination. Predicted sizes of RyR1, RyR2, and RyR3 were 224, 265, and 431 bp, respectively. No product was found if reverse transcriptase was omitted (No RT). (C) ATP (10 礛) induced increases in [Ca²⁺]_i in a cell before and after treatment with caffeine (10 mM).