**3. Results**

#### **3.1 Evidence for the presence of NHE1, NCX1, and Na/K ATPase**

#### *3.1.1 Sodium flux studies*

22Na uptake was measured in membrane vesicles from brain capillary endothelial cells, and revealed both saturable and unsaturable components (**Figure 1**). In the presence of specific inhibitors, three transport pathways were found, including: (a) a low affinity (Km = 52 mM), high capacity, dimethyl amiloride (DMA, 100 μM) sensitive sodium carrier, indicative of Na/H antiport; (b) a high affinity (Km = 4.6 mM), low capacity, DMA resistant carrier; and (c) a non-specific phenamil inhibitable cationic channel (Kd = 1.7 μl/mg/min).

#### *3.1.2 Immunoblotting*

Immunoblotting using specific antibodies (Chemicon) to NHE1 (Na/H antiporter, isoform 1) and NCX1 (Na/Ca exchanger, isoform 1) revealed that both proteins are present in isolated luminal and abluminal membranes derived from

#### **Figure 1.**

*Both saturable and unsaturable 22Na uptake was measured in plasma membrane vesicles derived from bovine brain capillary endothelial cells. A saturable component (Km = 52 mM) was dimethyl amiloride (DMA) sensitive (100 μM), indicative of a sodium-hydrogen ion exchanger.*

**203**

**Figure 2.**

*Prevention of Oxidative Injury Associated with Thrombolysis for Ischemic Stroke*

reported in the current study confirmed that it is the α3 isoform.

**3.2 Evidence that Na/H exchange elevates intracellular sodium**

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

exchange contributed to the sustained elevation of calcium.

*homology (96.8%) for the respective isoform in human tissue.*

brain capillary endothelial cells. NHE1 appeared as a single band with a molecular weight of 106 kD. NCX1 displayed the usual two bands at 43 kD and 150 kD.

Messenger RNA was prepared from isolated bovine brain capillaries, and RT-PCR was performed with specific primers identifying NHE1, Na/K ATPase α2, and Na/K ATPase α3. The expected PCR products were found for NHE1 (**Figure 2**) and Na/K ATPase α3, and sequencing demonstrated a high level of homology to the human transporters (>90%). Na/K ATPase activity was previously measured by us in luminal and abluminal membrane vesicles derived from endothelial cells forming the blood-brain barrier [25]. Activity was found to be present predominantly at the abluminal membrane domain, and was characterized by a ouabain binding constant (Kd = 25 ± 3 nM) typical of either the α2 or α3 isoform [49]. The PCR studies

Intracellular sodium concentration was measured in cultured bovine brain capillary endothelial cells exposed to conditions simulating ischemia and reperfusion. **Figure 3A** shows that intracellular sodium was elevated (P < 0.05) during ischemia (120 minutes), and remained elevated during ischemia followed by reperfusion (90/30 minutes). Inhibiting the Na/H antiporter with 100 μM dimethyl amiloride (DMA) completely prevented the elevation of intracellular sodium observed during ischemia/reperfusion (90/30 minutes), indicating that its activity was responsible for the sustained increase in intracellular sodium observed during

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

*The RT-PCR product for NHE1 was derived from bovine brain capillary endothelial cells, with a high* 

*DOI: http://dx.doi.org/10.5772/intechopen.84774*

*3.1.3 PCR studies*

ischemia and reperfusion.

brain capillary endothelial cells. NHE1 appeared as a single band with a molecular weight of 106 kD. NCX1 displayed the usual two bands at 43 kD and 150 kD.
