**3.3 Evidence that Na/Ca exchange elevates intracellular calcium**

Intracellular calcium was measured in cultured bovine blood-brain barrier cells exposed to conditions simulating ischemia and reperfusion. **Figure 3B** shows that calcium was significantly (P < 0.05) elevated during ischemia (120 minutes), and remained elevated during ischemia followed by reperfusion (90/30 minutes). Inhibition of the reverse mode of Na/Ca exchange (20 μM KB-R7943) reduced (P < 0.05) the level of intracellular calcium observed following reperfusion. Calcium uptake was also significantly inhibited with DMA (100 μM, not shown), indicating that both activation of Na/H antiport and reverse movement of Na/Ca exchange contributed to the sustained elevation of calcium.

#### **Figure 2.**

*The RT-PCR product for NHE1 was derived from bovine brain capillary endothelial cells, with a high homology (96.8%) for the respective isoform in human tissue.*

#### **Figure 3.**

*Simulating ischemia-reperfusion in cultured brain capillary endothelial cells resulted in a significant increase in intracellular sodium (panel a) during ischemia (120 minutes), that was maintained during ischemia followed by reperfusion (90/30 minutes). The rise observed during ischemia/reperfusion was prevented by inhibiting Na/H exchange (100 μM DMA). The same results were observed when measuring intracellular calcium (panel B). The rise in calcium during ischemia/reperfusion was significantly reduced with a specific inhibitor of the reverse movement of Na/Ca exchange (20 μM KB-R 7943). Intracellular calcium was also significantly reduced by DMA (100 μM (not shown). \*P < 0.05, different from control; + P < 0.05, different from ischemia/reperfusion. Values are mean ± SD. Measurements are made from 50 cells randomly chosen to represent each treatment.*
